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PubMed1994

Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B.

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)

Amino Acid Sequence↗
PubMed2004

Ca2+-calmodulin regulates fesselin-induced actin polymerization.

Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels.

Actins↗
PubMed1996

Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule.

We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.

Amino Acid Sequence↗
PubMed1997

Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains.

A fluorescent calmodulin derivative, 2-chloro-[4-(epsilon-amino-Lys75)]-[6-(4- diethylaminophenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin) [Török and Trentham (1994) Biochemistry 33, 12807-12820], and equilibrium fluorescence methods were used to identify calmodulin-binding domains of connexin subunits of gap junctions. Synthetic peptides corresponding to six extramembrane regions of connexin 32, a major component of rat liver gap junctions, and peptides derived from connexin 43 and 26, were tested. Two cytoplasmically oriented peptides that correspond to an N-terminal 21-amino-acid sequence and a 15-amino-acid sequence at the C-terminal tail of connexin 32 bound TA-calmodulin in a Ca2+-dependent manner. The dissociation constants (Kd) of TA-calmodulin binding to GAP 10 (MNWTGLYTLLSGVNRHSTAIG, residues 1-21) and GAP 8M (ACARRAQRRSNPPSR, residues 216-230) were 27 nM and 1.2 microM respectively at 150 mM ionic strength, 2 mM MgCl2, 100 microM CaCl2, pH 7.0 and 21 degrees C. Both halves of each peptide were required for calmodulin binding. Substitution of Trp3 present in all connexins by Tyr increased Kd for TA-calmodulin by 40-fold. Liver gap junctions (whose connexons contain mainly connexin 32) and recombinant connexons constructed of connexin 26 expressed by baculovirus-infected insect cells exhibited weaker binding of TA-calmodulin with variable Ca2+-dependence. These studies identify two calmodulin-binding amino-acid sequences in connexin 32, and provide independent evidence that calmodulin may function as an intracellular ligand, regulating Ca2+-dependent intercellular communication across gap junctions.

Amino Acid Sequence↗
PubMed2002

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor.

Previous work from our laboratory has demonstrated that the Ca(2+)-calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca(2+)-dependent calmodulin-affinity chromatography [San José, Bengurija, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237-15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645-660) binds calmodulin in a Ca(2+)-dependent manner when this segment forms part of a recombinant fusion protein [Martijn-Nieto and Villalobo (1998) Biochemistry 37, 227-236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca(2+), forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca(2+)-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K'(d)) of approx. 0.2-0.3 microM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.

Alkaline Phosphatase↗
PubMed1998

Calmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21(cip1).

One of the major signaling pathways by which extracellular signals induce cell proliferation and differentiation involves the activation of extracellular signal-regulated kinases (ERKs). Because calmodulin is essential for quiescent cells to enter cell cycle, the role of calmodulin on ERK2 activation was studied in cultured fibroblasts. Serum, phorbol esters, or active Ras induced ERK2 activation in NIH 3T3 fibroblasts. This activation was not inhibited by calmodulin blockade. Surprisingly, inhibition of calmodulin prior to fetal bovine serum addition prolonged activation of ERK2. Furthermore, inactivation of calmodulin in serum-starved cells induced ERK2 phosphorylation that was dependent on MAP kinase kinase (MEK). Inactivation of calmodulin in serum-starved cells also induced activation of Ras, Raf, and MEK. On the contrary, tyrosine phosphorylation of tyrosine kinase receptors was not observed. These results indicate that calmodulin inhibits ERK2 activation pathway at the level of Ras. Calmodulin inhibition induced overexpression of p21(cip1) which was dependent on MEK activity. We propose that inhibition of Ras by calmodulin prevents the activation of ERK2 at low serum concentration. Thus, entering into the cell cycle after serum addition would imply the overcoming of the inhibitory effect of calmodulin and consequently ERK2 activation. Furthermore, down-regulation of Ras by calmodulin may be also important to determine the duration of ERK2 activation and to prevent a high p21(cip1) expression that would lead to an inhibition of cell proliferation.

3T3 Cells↗
PubMed2005

Apoplastic calmodulin receptor-like binding proteins in suspension-cultured cells of Arabidopsis thaliana.

Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-3H]thymidine into nuclear DNA. Radioligand binding analysis with 35S-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the Kd approximately 9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with Kd values of approximately 47.5 and 830 nM. Chemical crosslinking of 35S-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.

Arabidopsis↗
PubMed1998

Characterization of microtubule binding domains in the Arabidopsis kinesin-like calmodulin binding protein.

The kinesin-like calmodulin binding protein (KCBP) is a new member of the kinesin superfamily that appears to be present only in plants. The KCBP is unique in its ability to interact with calmodulin in a Ca2+-dependent manner. To study the interaction of the KCBP with microtubules, we expressed different regions of the Arabidopsis KCBP and used the purified proteins in cosedimentation assays with microtubules. The motor domain with or without the calmodulin binding domain bound to microtubules. The binding of the motor domain containing the calmodulin binding region to microtubules was inhibited by Ca2+-calmodulin. This Ca2+-calmodulin regulation of motor domain interactions with microtubules was abolished in the presence of antibodies specific to the calmodulin binding region. In addition, the binding of the motor domain lacking the calmodulin binding region to microtubules was not inhibited in the presence of Ca2+-calmodulin, suggesting an essential role for the calmodulin binding region in Ca2+-calmodulin modulation. Results of the cosedimentation assays with the N-terminal tail suggest the presence of a second microtubule binding site on the KCBP. However, the interaction of the N-terminal tail region of the KCBP with microtubules was insensitive to ATP. These data on the interaction of the KCBP with microtubules provide new insights into the functioning of the KCBP in plants.

Animals↗
PubMed1988

Calmodulin-binding proteins in chromaffin cell plasma membranes.

Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10-fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins (240, 105, and 65 kilodaltons) were eluted by an EGTA-containing buffer. 125I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common (65, 60, 53, and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65-kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65-kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65-kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.

Adrenal Medulla↗
PubMed1987

Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis.

The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca2+/calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.

Calmodulin↗
PubMed1993

Calmodulin-binding proteins in bovine semen.

An 125I-labelled calmodulin gel overlay procedure was used to direct calmodulin-binding proteins in bovine spermatozoa and seminal plasma. Several calmodulin-binding proteins with molecular masses ranging from 12 to > 200 kDa were detected in epididymal and ejaculated spermatozoa. Certain of these proteins exhibited preferential calmodulin-binding in the presence of Ca2+, while others exhibited binding only in its absence. In seminal plasma, only two major proteins with molecular masses of 15 and 16 kDa showed a higher calmodulin-binding activity in the presence of Ca2+, whereas several polypeptides in the range of 6-17 kDa bound higher amounts of radiolabelled calmodulin in the absence of Ca2+. Our previous study has shown that a group of closely related major proteins, designated as BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa, isolated from bovine seminal plasma (BSP) have molecular masses in the range of 15-30 kDa. This prompted us to investigate whether these polypeptides from bovine seminal fluid interact with calmodulin. The results indicated that calmodulin binds to purified BSP-A1, -A2, -A3 and BSP-30 kDa proteins in the presence and absence of Ca2+. Furthermore, many polypeptides of low molecular mass (6-14 kDa) in bovine seminal plasma that crossreact with these BSP proteins also show high calmodulin-binding activity, particularly in the absence of calcium. This was further demonstrated following the limited proteolysis of the BSP proteins. Several tryptic-peptides of BSP-A1/-A2 and BSP-30 kDa exhibited higher calmodulin-binding activity than the intact BSP proteins. In view of the key role of Ca2+ in triggering the acrosome reaction and the role of calmodulin in intracellular transport of calcium, it is suggested that BSP proteins are involved in sperm capacitation and the acrosome reaction.

Animals↗
PubMed1983

Calmodulin-binding proteins in a cloned rat insulinoma cell line.

Calmodulin concentrations were measured in isolated hamster islets or in a cloned rat insulin-secreting cell line RIN-m-5F treated with high glucose. There was no change in the cellular calmodulin content of either islets or RIN-m-5F cells despite increases of insulin concentrations in the media. Treatment of cells with the anti-calmodulin drug W13 inhibited insulin-stimulated glucose release, whereas a small effect on insulin accumulation in the media was observed with W12, the dechlorinated, less active analogue of W13. At the lowest dose tested (30 microM) the effect of W13 on insulin accumulation in the media was completely reversible. To further investigate the possible role of calmodulin in insulin secretion, calmodulin-binding proteins in subcellular fractions of the RIN-m-5F cells were identified using a gel overlay technique. Ca2+-dependent binding of 125I-calmodulin was observed to cytosolic proteins with apparent Mr = 125, 110, 56, 52, and 34 k (kilodalton). This binding was completely displaceable with unlabeled calmodulin, whereas only partial displacement was observed when the homologous Ca2+ binding protein of skeletal muscle, troponin C, was used. Four proteins with Mr similar to the histones bind calmodulin in a Ca2+-independent manner. 125I-calmodulin:calmodulin binding protein interactions were inhibited in a dose-dependent manner by the anti-calmodulin drug, W13. Little effect was observed with the analogue W12.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma, Islet Cell↗
PubMed2001

A low-affinity Ca2+-dependent association of calmodulin with the Rab3A effector domain inversely correlates with insulin exocytosis.

The stimulus-response coupling pathway for glucose-regulated insulin secretion has implicated a rise in cytosolic [Ca2+]i as a key factor to induce insulin exocytosis. However, it is unclear how elevated [Ca2+]i communicates with the pancreatic beta-cell's exocytotic apparatus. As Rab3A is a model protein involved in regulated exocytosis, we have focused on its role in regulating insulin exocytosis. By using a photoactivatable cross-linking synthetic peptide that mimics the effector domain of Rab3A and microsequence analysis, we found calmodulin to be a major Rab3A target effector protein in pancreatic beta-cells. Coimmunoprecipitation analysis from pancreatic islets confirmed a Rab3A-calmodulin interaction in vivo, and that it inversely correlated with insulin exocytosis. Calmodulin affected neither GTPase nor guanine nucleotide exchange activity of Rab3A. The calmodulin-Rab3A interaction was pH- and Ca2+-dependent, and it was preferential for GTP-bound Rab3A. However, Rab3A affinity for calmodulin was relatively low (Kd = 18-22 micromol/l at 10(-5) mol/l [Ca2+]) and competed by other calmodulin-binding proteins that had higher affinity (e.g., Ca2+/calmodulin-dependent protein kinase-2 [CaMK-2] [Kd = 300-400 nmol/l at 10(-5) mol/l [Ca2+]]). Moreover, the Ca2+ dependence of the calmodulin-Rab3A interaction (K0.5 = 15-18 micromol/l [Ca2+], maximal at 100 micromol/l [Ca2+]) was significantly lower compared with that of the calmodulin-CaMK-2 association (K0.5 = 40 micromol/l [Ca2+], maximal at 1 mmol/l [Ca2+]). The data suggested that a transient Rab3A-calmodulin interaction might represent a means of directing calmodulin to the cytoplasmic face of a beta-granule, where it can be subsequently transferred for activation of other beta-granule-associated calmodulin-binding proteins as local [Ca2+]i rises to promote insulin exocytosis.

Animals↗
PubMed1994

Location of two contact sites between human smooth muscle caldesmon and Ca(2+)-calmodulin.

We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.

Actins↗
PubMed1986

Regulation of the calcium signal by calmodulin.

Stimulus-response coupling mediated by calmodulin involves several steps: a transitory increase in calcium concentration from 0.1 to 10 microM, induced by external stimuli; interaction of calcium with calmodulin, accompanied by stepwise structural transitions; the coordinated interaction with and activation of the many calmodulin-regulated enzymes and proteins. The binding of calcium to calmodulin is a cooperative and selective process that is modulated by magnesium. At physiological ionic strength, and only in the presence of magnesium, a large difference is seen between the affinities of sites III and IV (0.09 X 10(6) M-1) and sites I and II (0.0007 X 10(6) M-1) for calcium. This difference, together with the positive cooperativity previously observed, explains the stepwise conformational changes induced by calcium. The interaction of calmodulin with its target proteins requires the integrity of different portions of the calmodulin molecule. Calmodulin-regulated enzymes can be divided into three classes according to their abilities to bind with and to be activated by calmodulin fragments: enzymes which are activated by the C-terminal fragment, such as the Ca2+-ATPase and phosphorylase kinase; enzymes which require both halves of the molecule, such as cyclic AMP phosphodiesterase and myosin light chain kinase; and enzymes whose interaction with calmodulin fragments is too weak to be detected by activation, such as calcineurin and the multiprotein kinase. Thus different enzymes may be activated by different calmodulin conformers and the stepwise changes exhibited by calmodulin at different calcium levels can be used to regulate different metabolic pathways.

Animals↗
PubMed1996

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.

Amino Acid Sequence↗
PubMed1990

Erythrocyte Ca2(+)-ATPase: activation by enzyme oligomerization versus by calmodulin.

The subject of our studies is the mechanism of activation of the erythrocyte Ca2(+)-ATPase. Using purified, detergent solubilized enzyme it was found that equivalent maximal Ca2(+)-ATPase activity is obtained either upon addition of calmodulin or upon increase of enzyme concentration. Three independent methods, including Ca2(+)-ATPase activity, polarization of the enzyme modified with an external fluorescent probe, and efficiency of fluorescence resonance energy transfer between enzyme molecules have established that the concentration dependent activation is due to enzyme oligomerization. The oligomers bind calmodulin with a lower stoichiometry (0.5 mol calmodulin/mol Ca2(+)-ATPase), higher Ca2+ affinity (KCa = pCa 7.4), and higher cooperativity for Ca2+ (nH = 2.6) than the monomeric form (stoichiometry = 1 mol calmodulin/mol Ca2(+)-ATPase, KCa = pCa 7.0, nH = 1.1). The Ca2+ dependence of calmodulin binding and activation of monomers indicates that calmodulin binds before the Ca2(+)-ATPase activity is exhibited, demonstrating that the activation of this enzyme form is totally dependent on calmodulin. In contrast, oligomers reveal very similar Ca2+ dependence for calmodulin binding and for Ca2(+)-ATPase activity as well as for Ca2+ binding (assessed by tryptophan fluorescence), and for the oligomerization process (assessed by fluorescence energy transfer). The calmodulin antagonist drug 48/80 inhibits the calmodulin dependent activity of the monomers (I50 = 1.4 micrograms/ml) but has no effect on the activity of oligomers, confirming that calmodulin plays no role in the activation of the oligomeric enzyme. Our studies indicate that the erythrocyte Ca2(+)-ATPase can be activated by its high affinity, Ca2+ dependent binding of calmodulin or by a Ca2+ dependent oligomerization process which may involve calmodulin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1990

Protein kinase C inhibition by calmodulin and its fragments.

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10(-5) M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC50 of 6 microM compared to 1 microM of intact calmodulin. They were identified as Ser38-Arg74 and His107-Lys148. Each of the inhibiting fragments contains an intact Ca2(+)-binding domain with complete helix-loop-helix structure ("EF hand"). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.

Amino Acid Sequence↗