Phosphorylation of liver plasma membrane-bound calmodulin

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.     

Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.     

Calmodulin activity in yeast and mycelial phases of Ceratocystis ulmi

Abstract The yeast and mycelial phases of Ceratocystis ulmi contained roughly equivalent levels of calmodulin activity as determined by their ability to stimulate calmodulin-deficient bovine brain cAMP phosphodiesterase. This stimulation was calcium-dependent and could be inhibited by either dibucaine or trifluoperazine. Also, the concentration of dibucaine necessary to achieve the mycelium-to-yeast morphological conversion was found to be 3-fold greater in the presence of exogenous calcium. A model is presented in which only 30% of the cellular calmodulin need be complexed with calcium ions for mycelial development.     

Identification of calmodulin activity in purified retroviruses

Several viruses have been shown to require calcium for their function, and to bind calcium at specific sites. However, the nature of the calcium binding molecule on viruses has not been established. One possibility is the ubiquitous calcium-binding protein calmodulin. Our studies were designed to determine whether feline leukemia virus contained calmodulin. Accordingly, we tested purified feline leukemia virus for the presence of calmodulin-like activity. The virus, like authentic calmodulin, activated cyclic AMP phosphodiesterase. The ability of the virus to activate the enzyme was blocked in the presence of the known calmodulin inhibitors trifluoperazine and W-7. This indirect evidence for the presence of calmodulin was confirmed by radioimmunoassay. Several other retroviruses were also tested using radioimmunoassay and found to contain calmodulin. Our results indicate that the calcium binding site in retroviruses may be calmodulin.     

Interaction of calcium-binding proteins with calmodulin-dependent guanylate cyclase in Tetrahymena plasma membrane

Addition of bovine brain calmodulin and S-100 inhibited Tetrahymena calmodulin-induced stimulation of guanylate cyclase, but they did not affect enzymatic activity in the presence of calcium alone. Troponin C shows little effect on the cyclase activity regardless of the presence or absence of Tetrahymena calmodulin. The inhibitory effects of brain calmodulin and S-100 were overcome by the addition of Tetrahymena calmodulin, but not by calcium. Both calmodulins from Tetrahymena and bovine brain elicited stimulation of heart phosphodiesterase, while troponin C and S-100 did not affect the phosphodiesterase activity in the presence and absence of Tetrahymena calmodulin.     

Polyclonal antibody that recognizes calcium-dependent determinants in Tetrahymena calmodulin

We report here that a precipitating antibody prepared against Tetrahymena pyriformis calmodulin recognizes calcium-dependent determinants in the native protein. The ability of the antibody to precipitate 35S-labeled Tetrahymena calmodulin in direct radioimmunoassays was enhanced at least 3-fold in the presence of calcium. Competitive radioimmunoassay using homogeneous preparation of endogenously 35S-labeled Tetrahymena calmodulin and protein A-Sepharose-purified immunoglobulin G demonstrated that this antibody preparation is specific for protozoan calmodulin. Homogeneous vertebrate, invertebrate, and plant calmodulins, as well as rabbit skeletal muscle troponin C, did not show significant competition with the 35S-labeled Tetrahymena protein at concentrations 100-fold greater than that at which the homologous unlabeled Tetrahymena calmodulin produced 50% competition. A cyanogen bromide digest of Tetrahymena calmodulin also showed partial competition with the intact 35S-labeled protein, but only in the presence of calcium. The major antigenic determinants were localized to the carboxyl-terminal half of the molecule by immunoassay of limited trypsin fragments of Tetrahymena calmodulin. The antibody bound native calmodulin complexed to bovine brain phosphodiesterase (EC 3.1.4.17) but failed to recognize the Tetrahymena calmodulin carboxyl-terminal fragment (76-147) when complexed to the enzyme.     

Inhibition of mitosis in PtK2 cells by CAPP1-calmodulin

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.     

Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes

Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.     

Identification of testis specific calcineurin beta subunit isoform by a monoclonal antibody and detection of a specific six amino acid sequence.

Two isoforms of calcineurin beta subunit(beta 1 and beta 2) were identified in rat testis by a monoclonal antibody Va1. Both beta 1 and beta 2 were recovered in calmodulin binding protein fraction and showed calcium shift on SDS-polyacrylamide gel electrophoresis which is the specific character for EF-hand calcium binding protein. beta 2 showed same apparent molecular weight on SDS-PAGE as that of brain calcineurin beta and was found in wide variety of tissues. beta 1 was shown to have six amino acid polypepeptide sequence and it showed higher molecular weight than brain beta and was specific for testis.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

A calmodulin endoproteinase from mitochondrial membranes

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.     

Functional Heterogeneity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin-Dependent Protein Kinase II

Abstract: The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian α, β, γ, and δ isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher K _act for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower K _m for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1)

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.     

Calmodulin and the cell cycle: Involvement in regulation of cell-cycle progression

Calmodulin levels are elevated twofold at late G1 and/or early S phases during the growth cycle of CHO-K1 cells. These levels are maintained throughout the remainder of the cell cycle until cytokinesis. The G1 daughter cells then contain half the intracellular calmodulin level found prior to cell division. Elevation of calmodulin at the G1-S boundary is independent of the length of G1, and the increase in calmodulin appears to be related to progression into S phase. The importance of calmodulin for G1-S progression is suggested by the ability of the anticalmodulin drug W13 to elicit specific and reversible progression delays into and through S phase.     

A calcium binding protein from Drosophila melanogaster which activates cAMP phosphodiesterase: comparison of this protein with porcine brain calmodulin

A calcium binding protein from Drosophila melanogaster has been isolated and characterized. This protein shows several analogies with pig brain calmodulin in its molecular weight, isoelectric point, peptide maps, calcium binding properties, and ability to activate cyclic AMP phosphodiesterase. However, some differences were observed; the most remarkable one is the presence of tryptophan, an amino acid which is absent from all the calmodulins analyzed previously.     

NADPH analog binding to constitutive nitric oxide activates electron transfer and NO synthesis.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.     

Activation and stabilization of the catalytic unit of adenylate cyclase.

The requirements for stability and activity of the catalytic unit (C) of adenylate cyclase were investigated. After solubilization of bovine brain membranes in the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate (Chaps), the catalytic unit was separated from the stimulatory guanine-nucleotide-binding protein (Gs) by gel filtration on Ultrogel AcA-34. The partially purified C unit was rapidly inactivated at 30 degrees C; 0.25 mM-ATP stabilized activity. Although C-unit activity was dependent on Mg2+ or Mn2+, stabilization by ATP did not require bivalent cations. Activity of the Ultrogel-AcA-34-purified C unit was increased by Ca2+ plus calmodulin and by phosphatidylcholine plus lysophosphatidylcholine; activity in the presence of both activators was significantly greater than with each alone. Calmodulin plus Ca2+ and phospholipids also stabilized C unit. The column-purified C unit was activated by forskolin; the effect of forskolin was additive to those of calmodulin plus Ca2+ and phospholipids. p[NH]ppG-stimulated adenylate cyclase activity was reconstituted by mixing samples from the gel-filtration column containing Gs with C unit. Activation by Ca2+ plus calmodulin and Gs plus p[NH]ppG was additive; Ca2+ plus calmodulin did not alter the concentration of p[NH]ppG required for half-maximal activation. Results were similar with forskolin and Gs plus p[NH]ppG; the presence of one activator did not alter the effect of the other. These studies define conditions for separation of C unit and Gs from brain adenylate cyclase and demonstrate that ATP (in the absence of bivalent cations), phospholipids, calmodulin plus Ca2+, and forskolin all interact with C unit in a manner that is independent of functional Gs.     

Dynamics of calmodulin and cyclic AMP phosphodiesterase in plasma membranes of rat livers and ascites hepatomas

1. Plasma membranes from ascites hepatoma cells (AH-7974, AH-130) contained much smaller amounts of calmodulin (about half) and cyclic AMP phosphodiesterase (about one-third) compared to plasma membranes of rat livers. 2. Some of calmodulin molecules in liver plasma membranes were released by repeated washing. The 'washed' liver plasma membranes showed the presence of specific binding sites for externally added calmodulin molecules (bovine brain) (N = 140 pmol/mg protein, Kd = 7.9 . 10(-8) M). The calmodulin content of AH-7974 plasma membranes was not reduced by repeated washing. The binding of calmodulin to the 'washed' AH-7974 plasma membranes was only of nonspecific nature with negative cooperativity. 3. Plasma membranes (liver and AH-7974) appeared to contain both calmodulin-dependent and calmodulin-independent phosphodiesterase, but the stimulation by externally added Ca2+ plus calmodulin was rather small. Externally added calmodulin-dependent phosphodiesterase (bovine brain) was bound more to 'washed' liver plasma membranes than to 'washed' AH-7974 plasma membranes. Newly bound phosphodiesterase appeared to be more sensitive to the stimulation by Ca2+ plus calmodulin in 'washed' hepatoma plasma membranes than in 'washed' liver plasma membranes. 4. Preincubation of 'washed' plasma membranes (liver and hepatoma) with calmodulin did not affect the binding of phosphodiesterase, but the sensitivity of phosphodiesterase to the stimulation by Ca2+ plus calmodulin in hepatoma plasma membranes was lost.     

Inhibition of Calmodulin-Stimulated Phosphodiesterase Activity by Vasoactive Intestinal Peptide

The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin-dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 microM and 2 microM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 microM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 microM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.