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PubMed1981

Preparation of a fluorescent-labeled derivative of calmodulin which retains its affinity for calmodulin binding proteins.

Calmodulin was derivatized with 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid to fluorescently label the protein. This derivative (AEDANS.CaM) stimulated the Ca2+-sensitive cyclic nucleotide phosphodiesterase and formed Ca2+-dependent complexes with troponin I and the phosphodiesterase. Association between AEDANS.CaM and these proteins was directly monitored by changes in fluorescence anisotropy. The dissociation constants for the AEDANS.CaM--troponin I and AEDANS.CaM--phosphodiesterase complexes were 60 nM and 4 nM, respectively. This fluorescent derivative of calmodulin appears suitable for direct monitoring of the complexes between calmodulin and calmodulin binding proteins. Rotational diffusion of AEDANS.CaM was also measured with fluorescence anisotropy. These measurements indicated that the shape of calmodulin in solution is best approximated by a prolate ellipsoid.

Animals↗
PubMed1996

Inactivation of calmodulin-dependent protein kinase IV by autophosphorylation of serine 332 within the putative calmodulin-binding domain.

When brain calmodulin-dependent protein kinase IV is incubated with calmodulin-dependent protein kinase IV kinase under the phosphorylation conditions in the presence of Ca2+/calmodulin, rapid initial incorporation of 1 mol of phosphate into 1 mol of the enzyme by the action of the kinase kinase occurs, resulting in marked activation of the enzyme, and the subsequent incorporation of more than 3 mol of phosphate by autophosphorylation occurs, resulting in no significant change in the activity (Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116, 923-930; Okuno, S., Kitani, T., and Fujisawa, H. (1995) J. Biochem. (Tokyo) 117, 686-690). After the maximal phosphorylation, the continued incubation in the presence of excess EGTA resulted in additional autophosphorylation of the enzyme, leading to a complete loss of the Ca2+/calmodulin-dependent activity, while causing no significant change in the Ca2+/calmodulin-independent activity. The amino acid sequence analysis revealed that the autophosphorylation after removal of Ca2+ occurred on Ser332, Ser333, Ser337, and Ser341. Analysis by site-directed mutagenesis clearly showed that the autophosphorylation site responsible for the inactivation is Ser332. Thus, calmodulin-dependent protein kinase IV activated by the kinase kinase may lose its Ca2+/calmodulin-dependent activity by autophosphorylation on Ser332 located within the putative calmodulin-binding domain in the absence of Ca2+.

Amino Acid Sequence↗
PubMed1999

Brain myosin-V, a calmodulin-carrying myosin, binds to calmodulin-dependent protein kinase II and activates its kinase activity.

Myosin-V, an unconventional myosin, has two notable structural features: (i) a regulatory neck domain having six IQ motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized fraction of isolated nerve terminals. The purified proteins also coimmunoprecipitate from dilute solutions and bind in overlay experiments on Western blots. The binding region on myosin-V was mapped to its proximal and medial tail domains. Autophosphorylated CaM-kinase II binds to the tail domain of myosin-V with an apparent Kd of 7.7 nM. Surprisingly, myosin-V activates CaM-kinase II activity in a Ca2+-dependent manner, without the need for additional CaM. The apparent activation constants for the autophosphorylation of CaM-kinase II were 10 and 26 nM, respectively, for myosin-V versus CaM. The maximum incorporation of 32P into CaM-kinase II activated by myosin-V was twice that for CaM, suggesting that myosin-V binding to CaM-kinase II entails alterations in kinetic and/or phosphorylation site parameters. These data suggest that myosin-V, a calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II, a calmodulin-dependent enzyme.

Animals↗
PubMed1995

Paramecium Na+ channels activated by Ca(2+)-calmodulin: calmodulin is the Ca2+ sensor in the channel gating mechanism.

Paramecium Na+ channels, which were Ca(2+)-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10(-5) to 10(-3) M Ca+ in the bath. They became much less active in the presence of 10(-6) M Ca2+, and their activity subsided completely at 10(-8) M Ca2+. A Hill plot showed a dissociation constant of 6 microM for Ca2+ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mM Mg2+. The channels also exhibited a mild voltage dependence. When exposed to 10(-8) M Ca2+ for an extended period of 2-4 min, channels were further inactivated even after bath Ca2+ was restored to 10(-4) M. Whereas neither high voltage (+100 mV) nor high Ca2+ (10(-3) M) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca2+ to the bath restored channel activity without a requirement of additional Mg2+ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca2+ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na+ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na+ channel activation, and is the Ca2+ sensor of the Na(+)-channel gating machinery.

Animals↗
PubMed2000

Molecular evolution of calmodulin and calmodulin-like genes in the cephalochordate Branchiostoma.

Calmodulin is a calcium-binding EF-hand protein that is an activator of many enzymes as well as ion pumps and channels. Due to its multiple targets and its central role in the cell, understanding the evolutionary history of calmodulin genes should provide insights into the origin of genetic complexity in eukaryotes. We have previously isolated and characterized a calmodulin gene from the early-diverging chordate Branchiostoma lanceolatum (CaM1). In this paper, we report the existence of a second calmodulin gene (CaM2) as well as two CaM-like genomic fragments (CaML-2, CaML-3) in B. lanceolatum and a CaM2 and three CaM-like genes (CaML-1, CaML-2, CaML-3) in B. floridae. The CaM-like genes were isolated using low-stringency PCR. Surprisingly, the nucleotide sequences of the B. lanceolatum CaM1 and CaM2 cDNAs differ by 19.3%. Moreover, the CaM2 protein differs at two positions from the amino acid sequence of CaM1; the latter is identical to calmodulins in Drosophila melanogaster, the mollusc Aplysia californica, and the tunicate Halocynthia roretzi. The two B. lanceolatum CaM-like genes are more closely related to the CaM2 than to the CaM1 gene. This relationship is supported by the phylogenetic analyses and the identical exon/intron organization of these three genes, a relationship unique among animal CaM sequences. These data demonstrate the existence of a CaM multigene family in the cephalochordate Branchiostoma, which may have evolved independently from the multigene family in vertebrates.

Amino Acid Sequence↗
PubMed1989

Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase.

Measurements of ammonia release provide the first direct evidence that calmodulin becomes extensively deamidated during incubations at 37 degrees C, pH 7.4 or pH 11. A stoichiometry of 0.5 mol of NH3 released/mol of calmodulin is observed after 2 h at pH 11 or after 8-9 days at pH 7.4. These treatments also increase the ability of calmodulin to serve as a substrate for the isoaspartate-specific protein carboxyl methyltransferase from bovine brain. The stoichiometries of methylation are highly correlated with the stoichiometries of ammonia release. Deamidation and increased methyl-accepting capacity also occur in parallel for seven other proteins (aldolase, bovine serum albumin, cytochrome c, lysozyme, ovalbumin, ribonuclease A, and triosephosphate isomerase) upon incubation at pH 11. However, in comparison to calmodulin, these other proteins show very little deamidation and increased methylation capacity following incubation at pH 7.4. Deamidation of calmodulin at pH 7.4 is unaffected by the addition of 10(-7) M Ca2+; however, at 4 X 10(-6) M Ca2+, the rate of deamidation is inhibited by approximately 70%. The Ca2+-protection effect is consistent with the suggestion (B. A. Johnson, N. E. Freitag, and D. W. Aswad, (1985) J. Biol. Chem. 260, 10913-10916) that deamidation occurs preferentially at Asn-60 and/or Asn-97, each of which resides in a distinct Ca2+-binding domain.

Ammonia↗
PubMed1992

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.

Amino Acid Sequence↗
PubMed1982

Inhibition of calmodulin-activated cyclic nucleotide phosphodiesterase: multiple binding-sites for tricyclic drugs on calmodulin.

A cyclic nucleotide phosphodiesterase from guinea-pig heart is activated by calmodulin in the presence of calcium ions. Activation was measured over a range of calmodulin concentrations, and is antagonised by several tricyclic psychotropic drugs including trifluoperazine, imipramine, chlorpromazine and amitriptyline. When the concentration of amitriptyline was increased, its apparent inhibition constant for binding to calmodulin decreased. This was due in part to binding of amitriptyline to glass surfaces; but after correction for this the discrepancy was still significant. It is proposed that this is due to two sites on calmodulin for amitriptyline, with binding to either site being sufficient to prevent calmodulin from activating phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Full activation without calmodulin of calmodulin-dependent cyclic nucleotide phosphodiesterase by acidic glycosphingolipids: GM3, sialosylneolactotetraosylceramide and sulfatide.

Among calmodulin-non-binding glycosphingolipids, GM3, sialosylneolactotetraosylceramide (LM1), and sulfatide potently activated calmodulin-dependent cyclic nucleotide phosphodiesterase with or without Ca2+ showing ED50 1-5 microM. In contrast to calmodulin-binding gangliosides, these glycosphingolipids activated the enzyme up to the maximum level achieved by Ca2+/calmodulin and did not inhibit the activity at higher concentrations. Competition studies with GD1b that bind both to calmodulin and the enzyme suggest that the calmodulin-non-binding glycosphingolipids activate the enzyme through interaction with the same site of the enzyme as GD1b interacts.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis.

In response to water spray, subirrigation, wind, touch, wounding, or darkness, Arabidopsis regulates the expression of at least four touch-induced (TCH) genes. Ten to thirty minutes after stimulation, mRNA levels increase up to 100-fold. Arabidopsis plants stimulated by touch develop shorter petioles and bolts. This developmental response is known as thigmomorphogenesis. TCH 1 cDNA encodes the putative Arabidopsis calmodulin differing in one amino acid from wheat calmodulin. Sequenced regions of TCH 2 and TCH 3 contain 44% and 70% amino acid identities to calmodulin, respectively. The regulation of this calmodulin-related gene family in Arabidopsis suggests that calcium ions and calmodulin are involved in transduction of signals from the environment, enabling plants to sense and respond to environmental changes.

Air Movements↗
PubMed1996

Picosecond geminate recombination of CO to the complexes calmodulin*heme-CO and calmodulin*heme-CO*melittin.

Picosecond CO recombination kinetics have been measured after photodissociation of the artificial complexes calmodulin*heme-CO and calmodulin*heme-CO*melittin. These systems show an enhancement of the geminate fraction of kinetics relative to unbound heme-CO, due in part to fast geminate kinetics (tau=50ps for the initial phase), as well as a decrease in the rate of migration of CO away from the binding site. This indicates that calmodulin provides a complete pocket around the heme group. Rather than competing with the hemes for binding to calmodulin, the melittin seems to act as a cap to further enclose the hemes; melittin increases the affinity of calmodulin for heme-CO, but only weakly affects the CO recombination kinetics.

Amino Acid Sequence↗
PubMed1988

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain.

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.

Animals↗
PubMed2003

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin.

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.

Amino Acid Sequence↗
PubMed1996

Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calmodulin in fibroblast proliferation.

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 +/- 0.04 microgram/mmol creatinine) (P < 0.001), when compared to polycythaemia vera (PV) (0.14 +/- 0.02), essential thrombocythaemia (ET) (0.13 +/- 0.04), chronic myeloid leukaemia (CML) (0.16 +/- 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 +/- 0.02) and age-matched controls (0.1 +/- 0.02) (P < 0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age-matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS-induced proliferation of low-density fibroblasts but had little or no inhibitory effect on high-density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely, IFG-1, EGF, bDGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG-beta and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.

Adult↗
PubMed2004

Structure of potato calmodulin PCM6: the first report of the three-dimensional structure of a plant calmodulin.

The crystal structure of a potato calmodulin (PCM6) was solved by molecular replacement and refined to a crystallographic R factor of 22.8% (R(free) = 25.0%) using X-ray diffraction data in the resolution range 8.0-2.0 A. This is the first report of the three-dimensional structure of a plant Ca(2+)-calmodulin. PCM6 crystallizes in a crystal form that belongs to space group P2(1)2(1)2(1), which is different to that of most other calmodulin crystals. The main structural difference between PCM6 and the other calmodulins is in the central helix region and appears to be caused by crystal packing. The surface properties of PCM6 molecules were compared with those of animal calmodulins, which provided an explanation for the unique crystal-packing state of PCM6.

Amino Acid Sequence↗
PubMed1993

Interaction of calmodulin with a putative calmodulin-binding domain of inositol 1,4,5-triphosphate 3-kinase. Effects of synthetic peptides and site-directed mutagenesis of Trp165.

Recombinant rat brain inositol 1,4,5-triphosphate [Ins(1,4,5)P3] 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion product. It could be adsorbed onto calmodulin-Sepharose and eluted in Ca(2+)-free medium as a 48-kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin-binding domain on Ins(1,4,5)P3 3-kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin-binding domain. The inhibition was mimicked by a well-characterized peptide derived from the sequence of smooth muscle myosin light-chain kinase calmodulin-binding site. (b) The construction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.

Amino Acid Sequence↗
PubMed1989

Elevated calmodulin levels and reduced calmodulin-stimulated calcium-ATPase in Duchenne progressive muscular dystrophy.

We determined the calmodulin concentration and Ca2+-ATPase activity in subcellular fractions recovered from samples of vastus lateralis muscle obtained from 18 patients with Duchenne muscular dystrophy, 10 patients with other primary myopathies, 5 with spinal muscular atrophy, and 16 age-matched controls. Calmodulin levels were increased in the cytosol, plasmalemma, and heavy sarcoplasmic reticulum fractions from Duchenne dystrophy patients; the greatest increases occurred at early stages of disease or in mildly progressive cases. The total Ca2+-ATPase activities were decreased in the Duchenne dystrophy muscles; calmodulin caused a minimal stimulation of the activity in calmodulin-depleted membranes from Duchenne dystrophy compared with control membranes. The changes in calmodulin concentration and Ca2+-ATPase activity complement previous observations of reduced calsequestrin and dystrophin concentrations in Duchenne dystrophy muscles and suggest that these muscles lose calcium regulatory functions at early stages of the disease process.

Adenosine Triphosphatases↗
PubMed1988

1H-NMR studies of calmodulin: the modifying effect of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) on the calcium-induced conformational changes of calmodulin.

The effect of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin antagonist, on the calcium-bound conformation of calmodulin was studied by 1H-NMR at 400 MHz. W-7 affected the resonances of Ile-27, Phe-68, Phe-92, Ile-100, His-107 and Val-142. The resonances of Met-71, Met-72, Met-76, Phe-89 and Phe-141 may be affected by W-7. These findings suggest that W-7 binds to hydrophobic amino acid residues, which almost occur in calcium-binding sites II, III and IV or their vicinity. The effect of W-7 on the structure of calmodulin was similar to that of other drugs, trifluoperazine, D600 and oxmetidine. Thus, those residues in the high-field methyl region, the methionine methyl region and the phenylalanine aromatic region of calmodulin, which were similarly affected by all four drugs, may be important at the interface for binding of calmodulin to the regulatory sites on target enzymes.

Amino Acid Sequence↗