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A variety of proteins and tissue preparations (rabbit erythrocyte lysate, catalase, peroxidase, creatine phosphokinase, and lima bean trypsin inhibitor) contain protein activator(s) of the extracellular adenylate cyclase of intact Bordetella pertussis organisms. Stimulation of adenylate cyclase activity of up to 1000-fold over basal activity can be obtained. Activation of the adenylate cyclase is due to the presence of calmodulin in these protein preparations. The criteria to establish this were: Ca2+ dependence of the activation, inhibition by trifluoperazine, heat stability of the activator, chromatographic behavior like authentic calmodulin, and stimulation of cyclic nucleotide phosphodiesterase by the activators. The great sensitivity of the B. pertussis adenylate cyclase assay makes this and ideal system for the detection of trace amounts of calmodulin, in the presence of large amounts of other proteins.

Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity. CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated. Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis. Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM. The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter. In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity. As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation. This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state.

The 12th International Symposium on Calcium‐Binding Proteins and Calcium Function in Helth and Disease was held in February, 2002, in Cavalese, Italy. The 4‐day meeting consisted of nearly 60 plenary presentations and over 180 posters. ![][1] Our understanding of how calcium functions as a second messenger continues to increase at an incredible pace. This symposium covered most, if not all, aspects of the many roles of calcium in the regulation of cellular processes and ranged from calcium imaging through to the structure and function of calcium‐binding proteins. Emphasis was placed on elucidating the involvement of these proteins in the pathogenesis of a myriad of human diseases using genetic engineering technology. Unfortunately, so many interesting presentations were given that this review of the meeting can only cover a few topics that the authors feel resulted in conceptual advances in this broad field of science. We apologize to our many colleagues whose work is not given the coverage it deserves due to space limitations. ### Methodological advances It is fascinating to consider that the cell contains >1000 signaling proteins and only 10 second messengers. Calcium may be the most pervasive of the latter, as it can be spatially and temporally controlled with remarkable finesse and interacts with a dizzying array of proteins to execute its many regulatory functions. In response to signaling, calcium is transiently released into the cytosol from the external medium or the endoplasmic reticulum lumen and is sensed and modulated by a variety of calcium‐binding proteins. Calmodulin, which binds calcium due to its repeated ‘EF‐hand’ motif, is the most prominent member of this class of proteins because of its ability to activate a large number of target proteins, particularly those involved in protein phosphorylation, cyclic nucleotide metabolism and calcium homeostasis. T. Meyer (Stanford, CA) pointed out that analysis of the human genome predicts … [1]: /embed/graphic-1.gif

The solution structure of Ca2+-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca2+-ligated state. The difference between the crystal and solution structures of Ca2+–calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same χ1 rotameric states as in complexes with target peptide.

Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866].

The calmodulin-dependent phosphatase calcineurin is an important molecular target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Complexes of CsA and FK506 with their immunophilin ligands interact with calcineurin in vitro, resulting in the inhibition of its serine/threonine phosphatase activity toward polypeptide substrates. In order to demonstrate that CsA and FK506 inhibit calcineurin in vivo, we developed an assay to measure calcineurin phosphatase activity in crude cell extracts. We have previously reported that incubation of intact cells with nanomolar concentrations of either CsA or FK506 results in potent inhibition of calcineurin activity in a variety of cell lines. Here we discuss in detail the methodology and applications of the cell extract calcineurin assay.

The Ca2+-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimulated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca2+ and inhibited by the calmodulin-binding peptide, M13, and by the immunosuppressive drug, FK506. It is insensitive to rapamycin at concentrations up to 1 μM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction eluted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).

The dependence of calcineurin on Ca2+ for activity is the result of the concerted action of calmodulin, which increases the turnover rate of the enzyme and modulates its response to Ca2+ transients, and of calcineurin B, which decreases the Km of the enzyme for its substrate. The calmodulin-stimulated protein phosphatase calcineurin is under the control of two functionally distinct, but structurally similar, Ca(2+)-regulated proteins, calmodulin and calcineurin B. The Ca(2+)-dependent activation of calcineurin by calmodulin is highly cooperative (Hill coefficient of 2.8-3), and the concentration of Ca2+ needed for half-maximum activation decreases from 1.3 to 0.6 microM when the concentration of calmodulin is increased from 0.03 to 20 microM. Conversely, the affinity of calmodulin for Ca2+ is increased by more than 2 orders of magnitude in the presence of a peptide corresponding to the calmodulin-binding domain of calcineurin A. Calmodulin increases the Vmax without changing the Km value of the enzyme. Unlike calmodulin, calcineurin B interacts with calcineurin A in the presence of EGTA, and Ca2+ binding to calcineurin B stimulates native calcineurin up to only 10% of the maximum activity achieved with calmodulin. The Ca(2+)-dependent activation of a proteolyzed derivative of calcineurin, calcineurin-45, which lacks the regulatory domain, was used to study the role of calcineurin B. Removal of the regulatory domain increases the Vmax of calcineurin, as does binding of calmodulin, but it also increases the affinity of calcineurin for Ca2+. Ca2+ binding to calcineurin B decreases the Km value of calcineurin without changing its Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with I5N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (lH, 13C, and l5N). This assignment was verified by a 4D HN(C0CA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13Ca and 13C@ secondary chemical shifts, J(HNHa) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D I3C/ 15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loophelix (Kretsinger, R. H. (1 980) CRCCrit. Rev. Biochem. 8,119-1 741 calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.

The solution structure of Ca2+ ligated calmodulin and of its complex with a 26-residue peptide fragment of skeletal muscle myosin light chain kinase (skMLCK) have been investigated by multi-dimensional NMR. In the absence of peptide, the two globular domains of calmodulin adopt the same structure as observed in the crystalline form. The so-called 'central helix' which is observed in the crystalline state is disrupted in solution. 15N relaxation studies show that residues Asp78 through Ser81, located near the middle of this 'central helix', form a very flexible link between the two globular domains. In the presence of skMLCK target peptide, the peptide-protein complex adopts a globular ellipsoidal shape. The helical peptide is located in a hydrophobic channel that goes through the center of the complex and makes an angle of approximately 45 degrees with the long axis of the ellipsoid.

The three-dimensional solution structure of the complex between calcium-bound calmodulin (Ca(2+)-CaM) and a 26-residue synthetic peptide comprising the CaM binding domain (residues 577 to 602) of skeletal muscle myosin light chain kinase, has been determined using multidimensional heteronuclear filtered and separated nuclear magnetic resonance spectroscopy. The two domains of CaM (residues 6 to 73 and 83 to 146) remain essentially unchanged upon complexation. The long central helix (residues 65 to 93), however, which connects the two domains in the crystal structure of Ca(2+)-CaM, is disrupted into two helices connected by a long flexible loop (residues 74 to 82), thereby enabling the two domains to clamp residues 3 to 21 of the bound peptide, which adopt a helical conformation. The overall structure of the complex is globular, approximating an ellipsoid of dimensions 47 by 32 by 30 angstroms. The helical peptide is located in a hydrophobic channel that passes through the center of the ellipsoid at an angle of approximately 45 degrees with its long axis. The complex is mainly stabilized by hydrophobic interactions which, from the CaM side, involve an unusually large number of methionines. Key residues of the peptide are Trp4 and Phe17, which serve to anchor the amino- and carboxyl-terminal halves of the peptide to the carboxyl- and amino-terminal domains of CaM, respectively. Sequence comparisons indicate that a number of peptides that bind CaM with high affinity share this common feature containing either aromatic residues or long-chain hydrophobic ones separated by a stretch of 12 residues, suggesting that they interact with CaM in a similar manner.

The multiple functions of calmodulin in brain bring to light an apparent paradox in the mechanism of action of this multifunctional regulatory protein: How can the simultaneous calmodulin stimulation of enzymes with opposing functions, such as cyclic nucleotide phosphodiesterases and adenylate cyclase, which are responsible for the degradation and synthesis of cAMP, respectively, be physiologically significant? The same question applies to the simultaneous activation of protein kinases (in particular calmodulin kinase II) and a protein phosphatase (calcineurin). One could propose that the protein kinase(s) and the phosphatase may be located in different cells or in different cellular compartments, and are therefore not antagonizing each other. The same result could be achieved if the specific substrates of these enzymes have different cellular localizations. This does not seem to be the case. In many areas of the brain the two enzymes and their substrates coexist in the same cell. For example, the hippocampus is rich in calmodulin kinase II, calcineurin and substrates for the two enzymes. A more general scheme is presented here, based on different mechanisms of the calmodulin regulation of the two classes of enzyme, which helps to solve this apparent inconsistency in the mechanism of action of calmodulin.

The cloning and sequence determination of cDNAs encoding different types of serine/threonine protein phosphatases has provided a molecular basis for the protein phosphatase classification proposed by Ingebritsen and Cohen. Each of the phosphatases, phosphatase-1, -2A, -2B and -2C, exists as multiple isozymes raising the possibility that isozymes selectively expressed in different tissues may perform specific functions. The recent discovery of potent toxin inhibitors specific for protein phosphatase-1 and -2A will undoubtedly play an important role in the elucidation of the role of these enzymes in neuronal function.

New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an alpha-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been used: uniform labeling with 15N and labeling of specific amino acids with either 15N or 13C. It is demonstrated that uniform 15N labeling permits the recording of sensitive three-dimensional (3D) NMR spectra that show far better resolution than their conventional two-dimensional analogs. Selective 15N labeling of amino acids can be used for identifying the type of amino acid, providing information that is essential for the analysis of the 3D spectra. Simultaneous selective labeling with both 15N and 13C can provide a number of unique backbone assignments from which sequential assignment can be continued.

Kelvin J. A. Davies,* Gennady Ermak,* Beverley A. Rothermel, Melanie Pritchard, Joseph Heitman, Joohong Ahnn, Flavio Henrique-Silva, Dana Crawford, Silvia Canaider,** Pierluigi Strippoli,** Paolo Carinci,** Kyung-Tai Min, Deborah S. Fox, Kyle W. Cunningham, Rhonda Bassel-Duby, Eric N. Olson, Zhuohua Zhang, R. Sanders Williams, Hans-Peter Gerber,*** Merce Perez-Riba, Hisao Seo, Xia Cao, Claude B. Klee, Juan Miguel Redondo, Lois J. Maltais, Elspeth A. Bruford, Sue Povey, Jeffery D. Molkentin,**** Frank D. McKeon, Elia J. Duh, Gerald R. Crabtree,§§§§ Martha S. Cyert, Susana de la Luna, and Xavier Estivill

Previous studies have claimed that there is a failure of a mutant form of superoxide dismutase (mSOD) to protect the protein phosphatase, calcineurin (CN), against inactivation in the pathogenesis of amyotrophic lateral sclerosis (ALS), as determined in a murine model of ALS resulting from overexpression of mSOD (G93A). In contrast to previous studies, we find that mice overexpressing G93A mSOD have no statistically significant differences in the expression, or activity, of CN. However, CN from G93A mSOD overexpressing mice is significantly more protected against inactivation than non-transgenic mice that do not overexpress SOD. This reduced inactivation of CN is a consequence of increased expression of G93A mSOD. Thus, like wild-type SOD, G93A mSOD protects CN against inactivation.

Publisher Summary This chapter observes the regulation of calcineurin by oxidative stress. The protection of calcineurin against inactivation by superoxide dismutase and its reactivation by ascorbate, led to propose that inactivation was the result of oxidation of the iron cofactor. To identify the oxidation state of iron in the native enzyme, the purification procedure had to be modified to prevent the loss of the natural cofactors. Different assay conditions were used throughout the purification to monitor both the depletion of metal cofactors—requirement for metal ions in standard assays—and the oxidation state of iron—activation by ascorbate under anaerobic assay conditions. The reagents and procedure for calcineurin assays are discussed. The chapter describes the determination of calcineurin activity in crude tissue extracts. The protection of calcineurin against inactivation by superoxide dismutase suggested that the oxidation or reduction of calcineurin by superoxide anion (O − 2 ) was responsible for its inactivation. Accordingly, the rate of inactivation is increased in the presence of the superoxide generating agent, paraquat, or of xanthine and xanthine oxidase, and it is decreased under anaerobic conditions. Identification of calcineurin as an (Fe 2+ –Zn 2+ ) protein phosphatase is discussed.

Calcineurin (also called PP2B), a protein phosphatase under the control of Ca 2+ and calmodulin (CaM), is ideally suited to play an important role in modulating cellular responses in response to the second messenger Ca 2+ . The identification of calcineurin as the target of the immunosup-pressive drugs cyclosporin A (CsA) and FK506, complexed with their respective binding proteins cyclophilin A (CypA) and FKBP12 (FK506 binding protein), revealed the key role of calcineurin in the Ca 2+ -dependent steps of T-cell activation. This discovery led to purification of the transcription factor NFAT (nuclear factor of activated T cells) and the first identification of a complete transduction pathway from the plasma membrane to the nucleus. The specific inhibition of calcineurin by FK506 and CsA and the over-expression of the catalytic subunit of a CaM-independent derivative of calcineurin (calcineurin Aα, residues 1 to 392) have been widely used to identify the roles of calcineurin in the regulation of cellular processes as diverse as gene expression, ion homeostasis, muscle differentiation, embryogenesis, secretion, and neurological functions. It is no wonder that alteration of calcineurin activity has been implicated in the pathogenesis of such diseases as cardiac hypertrophy, congenital heart disease, and immunological and neurological disorders.

We have used site-directed mutagenesis, flow dialysis, and Fourier transform infrared (FTIR) spectroscopy to study Ca(2+)-binding to the regulatory component of calcineurin. Single Glu-Gln(E --Q) mutations were used to inactivate each of the four Ca(2+)-binding sites of CnB in turn, generating mutants Q1, Q2, Q3, and Q4, with the number indicating which Ca(2+) site is inactivated. The binding data derived from flow dialysis reveal two pairs of sites in the wild-type protein, one pair with very high affinity and the other with lower affinity Ca(2+)-binding sites. Also, only three sites are titratable in the wild-type protein because one site cannot be decalcified. Mutation of site 2 leaves the protein with only two titratable sites, while mutation of sites 1, 3, or 4 leave three titratable sites that are mostly filled with 3 Ca(2+) equiv added. The binding data further show that each of the single-site mutations Q2, Q3, and Q4 affects the affinities of at least one of the remaining sites. Mutation in either of sites 3 or 4 results in a protein with no high-affinity sites, indicating communication between the two high-affinity sites, most likely sites 3 and 4. Mutation in site 2 decreases the affinity of all three remaining sites, though still leaving two relatively high-affinity sites. The FTIR data support the conclusions from the binding data with respect to the number of titratable sites as well as the impact of each mutation on the affinities of the remaining sites. We conclude therefore that there is communication between all four Ca(2+)-binding sites. In addition, the Ca(2+) induced changes in the FTIR spectra for the wild-type and Q4 mutant are most similar, suggesting that the same three Ca(2+)-binding sites are being titrated, i.e., site 4 is the very high-affinity site under the conditions of the FTIR experiments.

Publisher Summary The use of the calcineurin inhibitors—namely, FK506 and CsA, together with yeast genetics and the overexpression of calcineurin by transgenic mice, has established the critical roles of calcineurin in the regulation of many cellular processes that are induced by changes in the concentration of intracellular Ca 2+ in response to external signals. None of the physiological roles of calcineurin is better documented than the regulation of gene expression mediated by the broadly distributed NFAT family of transcription factors in mammalian cells. Equally well documented is the role of calcineurin in the regulation of expression of many genes that are under the control of the TCNI/CRZl transcription factor in yeast. Calcineurim was originally identified as a major calmodulin-binding protein in the brain and later shown to be the only Ca 2+ /calmodulin-regulated serine/threonine protein phosphatase. Since then, this enzyme has been shown to be expressed in every tissue and to be highly conserved phylogenetically.

Over the past ten years the elucidation of the mechanism of action of the immunosuppressive drugs, cyclosporin A and FK506, has helped us to understand a major problem in biology at the molecular level: how are external signals acting on plasma membrane receptors transmitted to the nucleus. The specific inhibition by immunosuppressive drugs, complexed with their binding proteins, of the Ca2+/ calmodulin-dependent protein phosphatase, calcineurin, prevents the dephosphorylation and translocation to the nucleus of a family of transcription factors regulating the expression of many genes.

Calcineurin is the only protein phosphatase known to be under the control of Ca2+ and calmodulin. It is targeted by immunosuppressive drugs and has a critical role in T-cell activation. It is specifically inhibited by immunosuppressant immunophilin complexes, which enabled its function in regulating a wide range of cellular responses to Ca2+-mobilizing signals to be identified. Calcineurin in situ is 10-20 times more active than in the purified form and is subject to a time- and Ca2+/calmodulin-dependent reversible inactivation that is facilitated by small, heat-stable molecules. Here we identify a factor that prevents the inactivation of calcineurin in vitro and in vivo as the enzyme superoxide dismutase, which indicates that inactivation may be the result of oxidative damage to the Fe-Zn active centre of calcineurin. The redox state of iron provides a mechanism to regulate calcineurin activity by desensitizing the enzyme and coupling Ca2+-dependent protein dephosphorylation to the redox state of the cell. The protection of calcineurin against inactivation by superoxide dismutase constitutes a new physiological role for this enzyme which enables the Ca2+-dependent regulation of cellular processes to be modulated by the redox potential.

The three-dimensional structure of calmodulin in the absence of Ca2+ has been determined by three- and four-dimensional heteronuclear NMR experiments, including ROE, isotope-filtering combined with reverse labelling, and measurement of more than 700 three-bond J-couplings. In analogy with the Ca(2+)-ligated state of this protein, it consists of two small globular domains separated by a flexible linker, with no stable, direct contacts between the two domains. In the absence of Ca2+, the four helices in each of the two globular domains form a highly twisted bundle, capped by a short anti-parallel beta-sheet. This arrangement is qualitatively similar to that observed in the crystal structure of the Ca(2+)-free N-terminal domain of troponin C.

Calcineurin (CaN) is a Ca2+/calmodulin-dependent protein phosphatase found in brain and other tissues. It is a heterodimer consisting of a catalytic subunit (CaN-A) and a Ca(2+)-binding regulatory subunit (CaN-B). The primary structure of CaN-B indicates that it, like calmodulin, is an EF-hand protein and binds four Ca2+ ions. Eu3+, due to its favorable spectroscopic and chemical properties, has been substituted for Ca2+ in CaN-B to determine the metal ion-binding properties of this "calmodulin-like" protein. Excitation of the 7F0-->5D0 transition of Eu3+ results in a spectrum similar to that of calmodulin, consisting of three peaks. Analysis of the spectral titration curves reveals four Eu(3+)-binding sites in CaN-B. The affinities vary: sites I and II have dissociation constants of 1.0 +/- 0.2 and 1.6 +/- 0.4 microM, respectively; the values for sites III and IV are Kd = 140 +/- 20 and Kd = 20 +/- 10 nM, respectively. Binding of Tb3+ is slightly weaker. Tb3+ luminescence, sensitized by tyrosine, reveals that for lanthanides the highest affinity sites lie in the C-terminal domain. Energy transfer distance measurements between Eu3+ and Nd3+ in sites III and IV reveal a separation of 10.5 +/- 0.5 A, which suggests that these sites are arranged in a typical EF-hand pair. This information indicates that the overall structure of CaN-B is similar to the dumbbell-shaped proteins troponin-C and calmodulin, but is more like TnC in its metal-binding properties.

38 (6-32 residues) enzymically phosphorylated synthetic peptides have been assayed as substrates for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (PP-2B) belonging to the family of Ser/Thr-specific enzymes but also active on phosphotyrosine residues. Many peptides reproduce, with suitable modifications, naturally occurring phosphoacceptor sites. While protein phosphatases 2A and 2C are also very active on short phosphopeptides, an extended N-terminal stretch appears to be a necessary, albeit not sufficient, condition for an optimal dephosphorylation, comparable to that of protein substrates, of both phosphoseryl and phosphotyrosyl peptides by calcineurin. This finding corroborates the view that higher-order structure is an important determinant for the substrate specificity of calcineurin. However, a number of shorter peptides are also appreciably dephosphorylated by this enzyme, their efficiency as substrates depending on local structural features. All the peptides that are appreciably dephosphorylated by calcineurin contain basic residue(s) on the N-terminal side. A basic residue located at position -3 relative to the phosphorylated residue plays a particularly relevant positive role in determining the dephosphorylation of short phosphopeptides. Acidic residue(s) adjacent to the C-terminal side of the phosphoamino acid are conversely powerful negative determinants, preventing the dephosphorylation of otherwise suitable peptide substrates. However, calcineurin displays an only moderate preference for phosphothreonyl peptides which are conversely strikingly preferred over their phosphoseryl counterparts by the other classes of Ser/Thr-specific protein phosphatases. Moreover calcineurin does not perceive as a strong negative determinant the motif Ser/Thr-Pro in peptides where this motif prevents dephosphorylation by the other classes of Ser/Thr protein phosphatases. Whenever tested on phosphotyrosyl peptides, calcineurin exhibits a specificity which is strikingly different from that of T-cell protein tyrosine phosphatase, a bona fide protein tyrosine phosphatase. In particular while the latter enzyme is especially active toward a number of phosphopeptides reproducing the phosphoacceptor sites of src products and of calmodulin whose N-terminal moieties are predominantly acidic, the artificial substrate phospho-angiotensin II, bearing an arginine residue at position -2, is far preferred by calcineurin over all phosphotyrosyl peptides of similar size. Collectively taken these results show that the specificity of calcineurin, rather than resting on a given consensus sequence, is determined by a variety of primary and higher-order structural features conferring to it an overall selectivity that is different from those of any other known protein phosphatase.

At the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of approximately 25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to 15N or 13C are removed by isotope filtering and purge pulses.

Migration of human polymorphonuclear neutrophils on vitronectin is dependent on repeated transient increases in the concentration of intracellular free calcium ([Ca2+]i). A specific peptide inhibitor of the Ca(2+)-calmodulin-dependent phosphatase calcineurin was introduced into the cytoplasm of neutrophils. The peptide inhibited neutrophil migration on vitronectin by interfering with the release of the cells from sites of attachment. A similar reduction in motility on vitronectin occurred when cells were treated with the immunosuppressant FK506, which also inhibits calcineurin when bound to its binding protein, FKBP. These results indicate that a rise in [Ca2+]i reduces integrin-mediated adhesion to vitronectin by a mechanism that requires calcineurin activity.

The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.

The rate of proteolysis of trypsin-sensitive bonds was used to examine the nature of the structural changes accompanying Ca2+ and Mg2+ binding to calmodulin. In the Ca(2+)-free form, the rates of proteolysis at Arg-106 and Arg-37 are rapid (greater than 300 and 28 nmol min-1 mL-1, respectively), the bonds at Arg-74, Lys-75, and Lys-77, in the central helix, are cleaved more slowly (10 nmol min-1 mL-1), and a lag in the cleavage at the remaining bonds (Lys-13, Lys-30, Arg-86, Arg-90, and Arg-126) suggests that they are not cleaved in the native protein. High concentrations of Ca2+, but not Mg2+, almost completely abolish proteolysis at Arg-106 and drastically reduce the rate of cleavage at Arg-37. Both Ca2+ and Mg2+ exert a moderate protective effect on the proteolysis of the central helix. These results suggest that the F-helix of domains III and, to a lesser extent, the F-helix of domain I are somewhat flexible in the Ca(2+)-free form and are stabilized by Ca2+. Whereas full occupancy of the four Ca(2+)-binding sites produces little change in the susceptibility of the central helix to proteolytic attack, binding of two Ca2+ produces a 10-fold enhancement of the rate of proteolysis in this part of the molecule. We propose that at intermediate Ca2+ levels the flexibility of the central helix of calmodulin is greatly increased, resulting in the transient formation of intermediates which have not been detected by spectroscopic techniques but are trapped by the irreversible action of trypsin.

Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.

A novel Ca2+-binding protein (CBP-18) has been identified and purified from bovine brain. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein consists of a single band of apparent Mr 18,000 in the presence of Ca2+ or 20,000 in the presence of EGTA. CBP-18 contains one high affinity Ca2+-binding site, measured at 10(-5) M Ca2+ in the presence of 1 mM Mg2+ and 0.1 M K+. The amino acid composition and UV absorption spectrum distinguish CBP-18 from other Ca2+-binding proteins identified in brain. The protein has an extinction coefficient epsilon 1% 279 nm = 4.9 and contains 1 tryptophan/mol, 5 tyrosines/mol, and no trimethyllysine. CBP-18 does not interact with or activate calmodulin-stimulated phosphodiesterase. However, available evidence suggests that CBP-18 binds to other component(s) present in the brain extract in a Ca2+-dependent manner.

Phosphorylase kinase is of central importance in the nervous and hormonal control of glycogenolysis in mammalian skeletal muscle, since its activity is dependent on Ca*’ and stimulated by a phosphoryla- tion reaction catalysed by cyclic AMP-dependent protein kinase [ I]. Phosphorylase kinase from mammalian skeletal muscle has mol. wt 1 280 000 and possesses the struc- ture (@y8)4 where the o- and P-subunits are the components phosphorylated by cyclic AMP-dependent protein kinase [I] and the s-subunit is identical to the calcium binding protein termed calmodulin [2,3]. Calmodulin, formerly termed the modulator protein or calcium-dependent regulator protein, was originally identified as a factor which stimulated the activity of the high Km cyclic nucleotide phosphodiesterase of brain tissue [4,5] but it has subsequently been impli- cated in the control of a variety of intracellular processes which are regulated by Ca*’ [6-l 11. Calmodulin binds 4 Ca*‘/mol [ 121 and the calcium binding properties of phosphorylase kinase [ 131 support the view that all the high affinity binding sites for calcium are located on the 6-subunit [3]. This in turn suggests that the 6 -subunit is the compo- nent which confers calcium sensitivity to phosphorylase

Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.

Highly purified tryptic fragments of calmodulin were tested for their ability to stimulate adenylate cyclase activity of Bordetella pertussis spheroplast membranes and were compared to their activities on brain Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase. The C-terminal fragment, consisting of residues 78-148, was a full agonist for the cyclase with 0.1-0.15 the potency of calmodulin but did not stimulate phosphodiesterase. Fragments 1-77, 1-90, and 107-148 stimulated adenylate cyclase (and not phosphodiesterase) at low potency; this was not due to calmodulin contamination, but contamination by fragment 78-148 could not be excluded with certainty. An adduct of norchlorpromazine isothiocyanate and calmodulin showed full agonist activity for adenylate cyclase at 0.01-0.02 the potency of calmodulin. Stimulation of adenylate cyclase by a number of the fragments occurred in the absence of Ca2+, but stimulator potency was enhanced 20-60-fold in its presence. The similarity of Ca2+ requirements of fragment 78-148 and calmodulin suggests that occupancy of the two C-terminal Ca2+ binding sites of calmodulin accounts for most of the Ca2+ enhancement of calmodulin stimulation of adenylate cyclase.

The Ki for the interaction of 2-fluorourocanic acid with urocanase (from Pseudomonas fluorescens) is 1000 times as great as Km for the natural substrate, urocanic acid, whereas enzymatic hydration of the fluoro analog occurs ca. 100 times more slowly. Inhibition is competive and is eventually overcome by utilization of the analog. By contrast, 4-fluoro- and 2-amino-urocanic acid are neither significant inhibitors nor substrates for the enzyme. 2-Fluorourocanic acid may prove a useful tool for blocking the utilization of histidine as a one-carbon source in metabolism.

In affinity selection, calcineurin selects from a population of randomly modified calmodulins those species with which it prefers to interact. The method shows that acetylation of lysines affects calmodulin so as to interfere with its ability to interact with calcineurin. Monoacetylation of any lysine of calmodulin reduces its affinity for calcineurin by 5-10-fold. Multiple acetylations amplify the loss of affinity; none of the modifications are imcompatible with activity. The lack of selectivity of calcineurin against any particular modified lysine indicates that the loss of affinity reflects changes induced by the removal of the charged groups and suggests an important role for electrostatic interactions in the cooperative structural transitions which calmodulin undergoes upon binding its target proteins or calcium. In the presence of calcineurin, a large and specific decrease in the rate of acetylation of Lys-75 and -148 of calmodulin is observed. The reactivity of the same residues is greatly increased in the presence of calcium alone [Giedroc, D. P., Sinha, S. K., Brew, K., & Puett, D. (1985) J. Biol. Chem. 260, 13406-13413]. Lys-75, located in the central helix, and the C-terminal Lys-148 [Babu, Y. S., Sacks, J. S., Greenhouse, T. J., Bugg, C. E., Means, A. R., & Cook, W. J. (1985) Nature (London) 315, 37-40] may act as sensors of the calmodulin allosteric transitions. Their reactivity changes in opposite directions in response to calcium-induced or calcineurin-induced structural changes. The reactivity of other residues such as Lys-21, decreased in the presence of calcineurin but not calcium, is also affected by a conformational change which is induced specifically by calcineurin.

Limited proteolysis of calcineurin, the Ca2+/calmodulin-stimulated protein phosphatase, with clostripain is sequential and defines four functional domains in calcineurin A (61 kDa). In the presence of calmodulin, an inhibitory domain located at the carboxyl terminus is rapidly degraded, yielding an Mr 57,000 fragment which retains the ability to bind calmodulin but whose p-nitrophenylphosphatase is fully active in the absence of Ca2+ and no longer stimulated by calmodulin. Subsequent cleavage(s), near the amino terminus, yield(s) an Mr 55,000 fragment which has lost more than 80% of the enzymatic activity. A third, slower, proteolytic cleavage in the carboxyl-terminal half of the protein converts the Mr 55,000 fragment to an Mr 42,000 polypeptide which contains the calcineurin B binding domain and an Mr 14,000 fragment which binds calmodulin in a Ca2+-dependent manner with high affinity. In the absence of calmodulin, clostripain rapidly severs both the calmodulin-binding and the inhibitory domains. The catalytic domain is preserved, and the activity of the proteolyzed 43-kDa enzyme is increased 10-fold in the absence of Ca2+ and 40-fold in its presence. The calcineurin B binding domain and calcineurin B appear unaffected by proteolysis both in the presence and in the absence of calmodulin. Thus, calcineurin A is organized into functionally distinct domains connected by proteolytically sensitive hinge regions. The catalytic, inhibitory, and calmodulin-binding domains are readily removed from the protease-resistant core, which contains the calcineurin B binding domain. Calmodulin stimulation of calcineurin is dependent on intact inhibitory and calmodulin-binding domains, but the degraded enzyme lacking these domains is still regulated by Ca2+.

Calcineurin, a major calmodulin-binding protein of brain, is a heterodimer composed of a 61,000 Mr calmodulin-binding subunit, calcineurin A, and a 19,000 Mr Ca2+-binding subunit, calcineurin B. The discovery of a calmodulin-regulated protein phosphatase in rabbit skeletal muscle with a similar subunit structure led to the identification of calcineurin as a protein phosphatase (AA Stewart, TS Ingebritsen, A Manalan, CB Klee, P Cohen (1982) FEBS Lett 137:80-84). Using rabbit polyclonal antibodies to bovine brain calcineurin, both subunits of calcineurin can be identified in crude homogenates of bovine brain by an immunoblotting technique. In crude homogenates of bovine skeletal and cardiac muscle, a 59,000-61,000 Mr doublet and a 15,000 Mr species (the electrophoretic mobility of calcineurin B) are also detected by this technique. The cross-reactivity of these species with antibodies to brain calcineurin indicates antigenic similarity between the muscle proteins and calcineurin, and suggests the existence of a family of structurally related calmodulin-stimulated protein phosphatases. Like calcineurin, the 61,000 Mr subunits in skeletal and cardiac muscle bind calmodulin and are detected in crude tissue extracts by 125I-calmodulin gel overlay. Thus, both the 125I-calmodulin gel overlay method and the immunoblotting technique are useful in screening crude preparations, in which detection of calmodulin-stimulated protein phosphatase activity may be complicated by the many phosphatases present.