Bovine Calcineurin Research Articles

Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15-21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni(2+) was found to be the strongest stimulator, while Zn(2+) was found to inhibit CaN phosphatase activity. Mn(2+) was a relatively less effective stimulator compared to Ni(2+). Fe(2+) was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe(2+) slightly inhibited CaN activity from bovine brain. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.

A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants

The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Targeting of Calcineurin to an NFAT-like Docking Site Is Required for the Calcium-dependent Activation of the Background K+ Channel, TRESK

The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel) is activated in response to the calcium signal by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study we report that calcineurin also interacts with TRESK via an NFAT-like docking site, in addition to its enzymatic action. In its intracellular loop, mouse TRESK possesses the amino acid sequence, PQIVID, which is similar to the calcineurin binding consensus motif, PXIXIT (where X denotes any amino acids), necessary for NFAT (nuclear factor of activated T cells) activation and nuclear translocation. Mutations of the PQIVID sequence of TRESK to PQIVIA, PQIVAD, or PQAVAD increasingly deteriorated the calcium-dependent activation in the listed order and correspondingly reduced the benzocaine sensitivity (a property discriminating activated channels from resting ones), when it was measured after the calcium signal in Xenopus oocytes. Microinjection of VIVIT peptide, designed to inhibit the NFAT-calcineurin interaction specifically, also eliminated TRESK activation. The intracellular loop of TRESK, expressed as a GST fusion protein, bound constitutively active calcineurin in vitro. PQAVAD mutation as well as addition of VIVIT peptide to the reaction abrogated this calcineurin binding. Wild type calcineurin was recruited to GST-TRESK-loop in the presence of calcium and calmodulin. These results indicate that the PQIVID sequence is a docking site for calcineurin, and its occupancy is required for the calcium-dependent regulation of TRESK. Immunosuppressive compounds, developed to target the NFAT binding site of calcineurin, are also expected to interfere with TRESK regulation, in addition to their desired effect on NFAT.

Control of MEF2 Transcriptional Activity by Coordinated Phosphorylation and Sumoylation

A eukaryotic protein is often subject to regulation by multiple modifications like phosphorylation, acetylation, ubiquitination, and sumoylation. How these modifications are coordinated in vivo is an important issue that is poorly understood but is relevant to many biological processes. We recently showed that human MEF2D (myocyte enhancer factor 2D) is sumoylated on Lys-439. Adjacent to the sumoylation motif is Ser-444, which like Lys-439 is highly conserved among MEF2 proteins from diverse species. Here we present [corrected] several lines of evidence to demonstrate that Ser-444 of MEF2D is required for sumoylation of Lys-439. Histone deacetylase 4 (HDAC4) stimulated this modification by acting through Ser-444. In addition, phosphorylation of Ser-444 by Cdk5, a cyclin-dependent kinase known to inhibit MEF2 transcriptional activity, stimulated sumoylation. Opposing the actions of HDAC4 and Cdk5, calcineurin (also known as protein phosphatase 2B) dephosphorylated Ser-444 and inhibited sumoylation of Lys-439. This phosphatase, however, exerted minimal effects on the phosphorylation catalyzed by ERK5, an extracellular signal-regulated kinase known to activate MEF2D. These results identify [corrected] an essential role for Ser-444 in MEF2D sumoylation and reveal [corrected] a novel mechanism by which calcineurin selectively "edits" phosphorylation at different sites, thereby reiterating that interplay between different modifications represents a general mechanism for coordinated regulation of eukaryotic protein functions in vivo.

Protein Phosphatase 2B Dephosphorylates Twitchin, Initiating the Catch State of Invertebrate Smooth Muscle

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.

Activation of calcineurin expression in ischemia-reperfused rat heart and in human ischemic myocardium.

Calcineurin (CaN) has been reported as a critical mediator for cardiac hypertrophy and cardiac myocyte apoptosis. In the present study, we investigated the activity and expression of CaN and the effect of calpain in rat heart after ischemia and reperfusion. Rat ischemic heart showed significant increase in CaN activity. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine CaN indicated a prominent immunoreactive band of 60 kDa (CaN A). In ischemic-reperfused hearts, the expression of CaN A was significantly low and immunoreactivity was observed in proteolytic bands of 46 kDa. This may be due to the proteolytic degradation of CaN A in ischemic tissues by m-calpain. We also noticed in vitro proteolysis of bovine cardiac CaN A by m-calpain. Immunohistochemical studies showed strong staining of immunoreactivity in rat hearts that had gone under 30 min ischemia followed by 30 min reperfusion similar to that found in human ischemic heart. Ischemia is associated with multiple alterations in the extracellular and intracellular signaling of cardiomyocytes and may act as an inducer of apoptosis. The increase in CaN activity and strong immunostaining observed in ischemic/perfused rat heart may be due to the calpain-mediated proteolysis of this phosphatase.

Function and structure of recombinant single chain calcineurin

Calcineurin (CN) is a Ca 2+/calmodulin(CaM)-dependent serine/threonine protein phosphatase which is a heterodimer composed of a 61 kDa catalytic subunit (CNA) and a 19 kDa regulatory subunit (CNB). The enzyme is critical for several important intracellular signal-transducing pathways, including T-cell activation. Its crystal structure reveals that the C-terminal of CNB lies in close vicinity of the N-terminal of CNA and each end has a long arm not involved in the active site. After fusing two subunits, it was determined that folding and function of the protein were not affected by the fusion. We amplified a fused gene of A and B subunits using a pair of linker primers including six codons of glycine. A single chain calcineurin was constructed and purified to near-homogeneity. The recombinant enzyme was fully soluble, displayed high specific activity with substrate, and exhibited biochemical properties and kinetic parameters similar to those of the native enzyme from the bovine brain. It was still activated by Ca 2+/calmodulin but was not regulated by extra CNB and was still strongly stimulated by Mn 2+ and Ni 2+ divalent metal ions. The solution conformations of both recombinant enzyme and bovine calcineurin were assayed under the same conditions using intrinsic fluorescence spectroscopy and circular dichroism spectropolarimetry, and results showed their graphs are approximately identical. Our findings suggested that the fusion of A and B subunits of calcineurin does not affect their folding pathways and structural changes involved in their function, furthermore, they are bound to the correct binding site.

The Caenorhabditis elegans Homologue of Down Syndrome Critical Region 1, RCN-1, Inhibits Multiple Functions of the Phosphatase Calcineurin

A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. We have characterized Caenorhabditis elegans rcn-1, which belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demostrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1.

Characterization of cytosolic cyclophilin from guard cells of Vicia faba L.

The effect of immunosuppressant cyclosporin A (CsA) on inward-rectifying K(+)-channels and biochemical analysis have indicated the presence of cyclophilin in guard cells of Vicia faba. In this study, we identified a full-length cDNA sequence, vcCyP, encoding cyclophilin (CyP), a peptidyl-prolyl cis-trans isomerase of guard cell protoplasts (GCPs) from Vicia faba L. The deduced amino acid sequence revealed that vcCyP contained 171 amino acid residues and exhibited a strong similarity to previously described cytosolic CyP isoforms from other plants. vcCyP had seven extra amino acid residues, which is a characteristic of the cytosolic form of plant CyPs. A complex of recombinant vcCyP and CsA inhibited the phosphatase activity of bovine calcineurin, a type 2B protein phosphatase, with a half-inhibitory concentration of 0.2 microM. Protein phosphatase activity was measured in the cytosolic fraction of GCPs using a 32P-labeled myelin basic protein (32P-MBP) and the activity was increased by a physiological concentration of Ca2+ (1 microM). This Ca(2+)-stimulated phosphatase activity was inhibited by CsA, suggesting the presence of both cytosolic CyP and calcineurin-like protein phosphatase in guard cells. Northern blot analysis showed that the transcription level of vcCyP was much higher in GCPs than in root and leaf tissues of Vicia.

Roles of peptidyl-prolyl CIS-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin a, FK506, and rapamycin

The immunosuppressive peptide cyclosporin A inhibits the growth of malaria parasites in vitro and in vivo, but little is known about its mechanism of antimalarial action. The immunosuppressive action of cyclosporin A is believed to result from binding of the drug to cyclophilins (intracellular peptidyl-prolyl cis-trans isomerases), and inhibition of the protein phosphatase calcineurin by the cyclosporin A-cyclophilin complex. Two immunosuppressive macrolides, FK506 and rapamycin, bind to a distinct isomerase, FKBP12, and the FK506-FKBP complex also inhibits calcineurin. Calcineurin itself is apparently involved in signal transduction between the T-cell membrane and nucleus, and its inhibition blocks T-cell activation. Rapamycin inhibits a later step in T-cell proliferation. Peptidyl-prolyl cis-trans isomerase activity was detected in extracts of Plasmodium falciparum. It was completely inhibited by concentrations of cyclosporin A above 0.1μM, but not by FK506 or rapamycin, and probably represented one or more cyclophilins. Comparison of the antimalarial and anti-isomerase activities of a series of cyclosporin analogues failed to reveal a correlation between the two properties. Cyclosporin A and its more active 8'-oxymethyl-dihydro-derivative, in combination with the cyclophilin-containing P. falciparum extract, inhibited the protein phosphatase activity of bovine calcineurin. Therefore inhibition of a putative P.falciparum calcineurin by a complex of CsA and cyclophilin might be responsible for the antimalarial action of the drug. The most active cyclosporin, however, was a 3'-keto-derivativ of cyclosporin D (SDZ PSC-833) which inhibited P. falciparum growth with a 50% inhibitory concentration ( ic 50) of 0.032 μM (compared with 0.30 μM for cyclosporin A), but was a poor inhibitor of the parasite isomerase. 3'-Keto-cyclosporin D has negligible immunosuppressive activity, but it strongly inhibits the P-glycoprotein of multi-drug resistant mammalian tumour cells. FK506 and rapamycin were also active antimalarials ( ic 50 of 1.9 and 2.6mu;M, respectively) but in the absence of detectable FKBP in P.falciparum extracts, their mechanisms of antimalarial action remain unclear.

PCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophillins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells. Previously, we reported that higher plants also express immunophilins and have a Ca(2+)-dependent signaling pathway sensitive to immunophilin-ligand complexes. Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin. The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting. The mature pCyP B protein has rotamase activity with low-substrate specificity. Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM. Similar to other CyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin. The mRNA level of pCyP B was high in leaf tissue but was not detectable in roots. Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock. These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.

Overexpression and Characterization of a Recombinant Form of Rat Calcineurin A

Overexpression of rat recombinant calcineurin A catalytic subunit in E. coli was achieved using a system under control of the T7 promoter. The specific activity of the purified catalytic subunit was suppressed relative to native bovine calcineurin, with the extent of suppression depending upon the choice of substrate. Addition of calcineurin B subunit stimulated phosphatase activity to one third that of native calcineurin. The metal activators Mn 2+ and Ni 2+ as well as several anion inhibitors affected both native calcineurin and recombinant calcineurin A activity to the same extent. In addition, calcineurin B was required for inhibition by the immunosuppressive complex FK506-FK506-binding protein.

Isolation and characterization of a cDNA clone coding for the calcium-binding subunit of calcineurin from bovine brain: An identical amino acid sequence to the human protein

A cDNA clone encoding the calcium-binding subunit of calcineurin, calcineurin B, was isolated from a bovine brain library by immunoscreening. The 841 bp cDNA has a 56 bp 5'-noncoding region, an open reading frame of 510 bp, and a 275 bp 3'-noncoding sequence. The deduced amino acid sequence of bovine calcineurin B differs from the previously reported protein sequence (Aitken et al., 1984) by three residues. The sequence contained additional valine at the carboxyl terminus and substitutions of Met-11 and Ser-153 (the positions according to Aitken et al., 1984) by cysteine. The amino acid sequence of bovine calcineurin B was found to be identical to that of human calcineurin B sequence (Guerini et al., 1989). In fact, 97.1% homology was observed between the coding regions of human and bovine calcineurin B. In addition, a very high homology of 95.2% was observed for the 3'-noncoding region while the 5'-noncoding region showed 58.9% homology. The beta-galactosidase fusion protein, having the apparent molecular weight of 29 kDa, was detected on Western blots by subunit B-specific monoclonal antibody (Matsui et al., 1985). Northern analysis revealed that there is a single calcineurin B transcript in bovine brain which is 2.3 kb in length. This is in agreement with the observation of only one immunologically detectable subunit B protein in bovine brain (Matsui et al., 1985).

Binding of Active Cyclosporins to Cyclophilin A and B, Complex Formation with Calcineurin A

The binding properties of several active and inactive cyclosporins to the major intracellular receptor proteins, cyclophilin A and B, as well as the interaction with the phosphatase calcineurin were investigated by ELISA and by means of a photoaffinity labeled probe (PL-CS). Binding to recombinant human cyclophilin A and B was rapid and saturable, and correlated with the in vitro immunosuppressive activity of cyclosporin derivatives. In the presence of cyclophilin A or B and calcium cyclosporin binds specifically to purified bovine calcineurin. PL-CS labeled only the calcineurin A subunit, but not the B subunit or calmodulin. Calcineurin A binding was competed by active (CsA, CsG or CsM), but not inactive (CsH, CsF) derivatives or the structurally unrelated macrolide immunosuppressant FK506. Ternary complexes containing equimolar ratios of cyclophilin A or B, PL-CS and calcineurin were resolved by chemical-crosslinking. The formation of these complexes was apparently specific, calcium-, but not calmodulin-dependent, and only inhibited by active cyclosporins. In vivo labelling of Jurkat T-cells revealed, that cyclophilin A and calcineurin A are the main labeled proteins, which form complexes in the presence of active cyclosporin. Thus, we demonstrate directly, that active cyclosporins have two recognition sites, which allow the in vivo recognition of cyclophilins and calcineurin A.

FKBP12-FK506 complex inhibits phosphatase activity of two mammalian isoforms of calcineurin irrespective of their substrates or activation mechanisms.

The interaction of calcineurin (Ca2+/calmodulin-dependent protein phosphatase) with the potent immunosuppressive agent FK506 and its 12 kDa isoform binding protein (FKBP12) was investigated. The FKBP12-FK506 complex inhibited the Ca2+/calmodulin-stimulated phosphatase activity of each of two calcineurin isoforms, which contain either the catalytic subunit A alpha or A beta (calcineurin A alpha or A beta) of bovine calcineurin. Calcineurin phosphatase activity was inhibited by the FKBP12-FK506 complex irrespective of the substrate or the enzyme activation mechanism. FK506 and FKBP-12 inhibited calcineurin in a concentration-dependent manner, and complete inhibition of the phosphatase activity appeared to require a molar excess of FKBP12-FK506 complex. Immunochemical measurements revealed tissue differences in the concentration of calcineurin, which may be of importance to the selectivity for immunosuppression of all of the biological effects. Direct binding studies with [3H]dihydro-FK506 suggest that the ratio of FKBP12-FK506 complex to calcineurin in vivo when IL2 production is inhibited is well correlated with the ratio when calcineurin phosphatase activity is inhibited in vitro. These results suggest that calcineurin is a relevant cellular target of FK506 when bound to FKBP-12.

Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.

By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.

Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B.

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.

CDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae

We have isolated a cDNA clone encoding a homolog of mammalian calcineurin B (the regulatory subunit of calmodulin-dependent protein phosphatase) by screening a cDNA expression library of Saccharomyces cerevisiae with antiserum against bovine calcineurin B. The yeast calcineurin B homolog (YCNB) is composed of 175 amino acids with a calculated molecular mass of 19,639 daltons and contains four putative Ca(2+)-binding domains. The amino-acid alignment of YCNB with human calcineurin B demonstrates 53% sequence identity and 82% homology. Southern blot analysis indicates that the gene for YCNB is a single-copy gene. Thus, yeast calmodulin-dependent protein phosphatase apparently has a heterodimeric structure similar to that of the enzyme in mammalians.

Formula omitted] protein phosphatase in bovine parotid gland: Purification and characterization

Calmodulin-dependent protein phosphatase (CaM-PPase) was isolated from bovine parotid gland by sequential application of DEAE-52, Affi-gel blue and calmodulin-affinity chromatography followed by gel filtration and high performance liquid chromatography. The enzyme was activated in the simultaneous presence of Ni 2+ or Mn 2+ and Ca 2+ plus calmodulin. Ca 2+ calmodulin -dependent activation of CaM-PPase was antagonized by inhibitors of calmodulin action, such as W-7 and trifluoperazine. Tryptophan fluorescence was quenched in the presence of Ni 2+. CaM-PPase was a heterodimer. The molecular weights of large subunits which bound calmodulin (CaM) were 68 kD and 58 kD — the 68 kD subunit was predominant. Polyclonal antibodies against bovine calcineurin cross-reacted with both types of larger subunits. Using polyclonal antibodies against bovine calcineurin or the monoclonal antibody against subunit B of bovine calcineurin, the smaller molecular weight subunit (19 kD) was found to be immunologically identical to subunit B of bovine calcineurin. In bovine parotid gland, CaM-PPase was found both in acinar and duct cells.

Effects of modifying individual amino or carboxyl groups on the affinity of calmodulin for calcineurin

The effects of modifying individual lysyl, aspartyl, or glutamyl residues in calmodulin on its ability to bind to the neural phosphatase calcineurin have been investigated using a competitive binding method termed "label selection." Samples of calmodulin were radiochemically labeled at a low level (0.03-0.6 group/molecule) by acetylation of amino groups or coupling carboxyl groups with ethanolamine to produce preparations containing predominantly single-site modified and unmodified molecules. These preparations were incubated in a 5-10-fold molar excess with bovine calcineurin under conditions appropriate for complex formation. The bound population was isolated, and the level of modification of each reactive residue was compared with the level in the corresponding group in the intial unselected preparation to determine if molecules modified at specific sites had been selected for or against during the competition for complex formation. Significant selection was observed against molecules modified at Lys21, Asp64, Glu67, Lys75, Glu84, Glu114, Asp118, or Lys148, whereas modification of Glu83 increased binding. The modification of other groups, including components of the four Ca2+-binding sites, had no effect on the interaction. Glu67, a Ca2+-liganding residue in Ca2+-binding site II that may regulate the orientation of this site in relation to the central helix, had the strongest influence on complex formation. Most of the residues identified form a nearly linear array in the three-dimensional structure of calmodulin and indicate the location of an extended surface for interaction with calcineurin and other enzymes.