TY - JOUR. T1 - Fluorescence analysis of calmodulin mutants containing tryptophan. T2 - Conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II. AU - Prendergast, Franklyn G.. PY - 1991. Y1 - 1991. N2 - Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of ...
Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca2+ dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (Kact 1.8 and 1.7nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher Kact than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca2+-ATPases. The plant Ca2+-ATPase was activated maximally by both isoforms, while the erythrocyte Ca2+-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase ...
Techniques such as X-ray structural analysis offer snapshots, at best, of steps in this intracellular work flow. But single-molecule atomic-force spectroscopy has opened a new window on such dynamic processes.. Professor Matthias Rief and colleagues at the Technische Universitaet Muenchen had previously shown that they could fix a single calmodulin molecule between a surface and the cantilever tip of a specially built atomic-force microscope, expose it to calcium ions in solution, induce peptide binding and unbinding, and measure changes in the molecules mechanical properties as it did its work. "What is special about our technique," Rief says, "is that we can work directly in aqueous solution. We can make our measurements in exactly the conditions under which the protein works in its natural environment. So we can directly observe how the calmodulin snatches the amino acid chain and folds itself, to hold its target fast." Measuring the force needed to bend the calmodulin molecule out of its ...
Elsewhere, we have reported the structure of a rat calmodulin gene and two distinct rat calmodulin cDNAs, pRCM1 and pRCM3. Here, I report the cloning and sequencing of the third calmodulin cDNA (pRCM4) and two additional rat calmodulin genes. The original calmodulin gene is named CaM I (pRCM1) and t …
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N-methyl-D-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membraneproximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875-1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca²⁺/calmodulin binds GluN2A with high affinity (5.2 ± 2.4 nM) in vitro. Direct interaction of Ca²⁺/calmodulin with GluN2A was not affected by disruption of classic sequence motifs ...
Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses Published In Frontiers in Plant Science, 6:600, 2015, by Houqing Zeng, Luqin Xu, Amarjeet Singh, Huizhong Wang, Liqun Du, B. W. Poovaiah ...
TY - JOUR. T1 - Calmodulin binds to the C terminus of sodium channels Nav1.4 and Navl.6 and differentially modulates their functional properties. AU - Herzog, Raimund I.. AU - Liu, Chuanju. AU - Waxman, Stephen G.. AU - Cummins, Theodore R.. PY - 2003/9/10. Y1 - 2003/9/10. N2 - Modulation of voltage-gated sodium channels (VGSC) can have a major impact on cell excitability. Analysis of calmodulin (CaM) binding to GST-fusion proteins containing the C-terminal domains of Navl.l-Na vl.9 indicates that some of the tetrodotoxin-sensitive VGSC isoforms, including Navl.4 and Navl.6, are able to bind CaM in a calcium-independent manner. Here we demonstrate that association with CaM is important for functional expression of Nav1.4 and Navl.6 VGSCs. Disrupting the interaction between CaM and the C terminus of Na vl.4 and Navl.6 channels reduced current amplitude by 99 and 62%, respectively. Overexpression of CaM increased the current generated by Navl.4 and Navl.6 C-terminal mutant constructs that ...
The calcium-signaling network is an important transducer of internal and external stimuli in plants. These signals are transduced by a divers set of calcium-sensors such as calmodulin and calmodulin like proteins. In this work, AFG1L2 (AFG1 like protein 2) was characterized as a calmodulin binding protein that belongs to the family of AAA+ proteins (ATPases associated with various cellular activities). Using GFP fusion constructs it was possible to determine the in vivo localization of AFG1L2 in mitochondria and also to confirm the dual localization of a previously described homologe, AFG1L1, to mitochondria and chloroplasts. The interaction between AFG1L2 and calmodulin is calcium dependent and the calmodulin binding site of the AFG1L2 protein is in the AAA domain at a site homologous to the AFG1L1 protein, in very close proximity to the Walker A Motif, which is essential for ATP hydrolysis. It could be shown that ATP and ADP intensify the interaction between AFG1L2 and calmodulin. AAA-proteins ...
Mutations: The following mutations were used: Camn339, recessive RNA null mutation (Heimanet al. 1996); Cam7, recessive ethyl methanesulfonate mutation (Nelsonet al. 1997); Cam352, recessive hypomorph, generated by excision of a P element in 5′ flanking DNA (Scottet al. 1997); Cam3909, recessive hypomorph generated by a P insertion 60 bp 5′ of the transcription start site (Harvieet al. 1998); Ryr16, recessive mutation of the ryanodine receptor gene (Ryr; Sullivanet al. 2000); Df(2R)H3E1, deficiency with breakpoints at 44D1-4 and 44F12 (Bloomington Stock Center); Ca-α1DX7, embryonic lethal Ca-α1D mutation (Eberlet al. 1998); Ca-α1DAR66, hypomorphic Ca-α1D mutation (Eberlet al. 1998; Renet al. 1998); and cn1, cinnabar (Bloomington Stock Center).. Gal4 lines: We used the following Gal4 lines: 24B-Gal4, P{GawB}how24B, an insertion into held out wings that expresses Gal4 in muscle (Brand and Perrimon 1993); elav-Gal4, Gal4 expressed under the elav promoter in neurons at all developmental ...
The regulation of the guinea-pig pancreatic acinar plasma membrane Ca2+ pump by protein kinase A, protein kinase C and calmodulin was investigated. The results were compared with the effects of these regulators on the high affinity Ca2+-ATPase found in this membrane preparation. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca2+ transport 2-fold, but had no effect on Ca2+-dependent ATPase activity. Purified protein kinase C, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate and diacylglycerol derivative, 1-stearoyl-2-arachidonoyl-sn-glycerol, failed to stimulate the Ca2+-uptake but augmented the Ca2+-dependent ATPase activity. Exogenously added calmodulin failed to stimulate either activity. In addition, two antagonists of calmodulin activity, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca2+-transport. These data suggest the presence of endogenous calmodulin within guinea-pig pancreatic acinar plasma membranes. Both calmodulin
Biological systems primarily use proteins to sense and respond to other molecules. We sought to engineer a protein-based platform as the basis of a biologically emulative biosensor. This biosensor platform is especially powerful if both the analyte sensitivity and transduction method can be straightforwardly customized to respond to important targets. Here we show our attempts to demonstrate the modularity of a pre-existing protein biosensor through such customization. The calcium-binding protein calmodulin has already been engineered to exhibit enzymatic switching in response to peptide binding through fusion to a split TEM1 ß-lactamase. To extend this platform, we first evolved the calmodulin-ß-lactamase fusion (BlaCaM) to exhibit sensitivity to a previously inactive peptide, Staphylococcus aureus ∂-toxin, through random mutagenesis of the calmodulin portion of BlaCaM, followed by screening the purified library for ß-lactamase activity. Second, we altered the transduction domain by ...
Calmodulin (CaM) (an abbreviation for CALcium MODULated proteIN) is a calcium-binding protein expressed in all eukaryotic cells. It can bind to and regulate a number of different protein targets, thereby affecting many different cellular functions. Calmodulin 1 is one of nearly twenty human calmodulins.Calmodulinis the archetype of the family of calcium-modulated proteins of which nearly 20 members have been found. They are identified by their occurrence in the cytosol or on membranes facing the cytosol and by a high affinity for calcium. Calmodulin contains 148 amino acids and has 4 calcium-binding motifs. Its functions include roles in growth and the cell cycle as well as in signal transduction and the synthesis and release of neurotransmitters.
Transcription factor regulating the cell cycle specific transcription of a spindle pole body (SPB) calmodulin binding protein SPC110. Required for full induction of SPC110 transcription in late G1. Binds to DNA consensus sequence 5-[AT]AA[TC]AAACAA[AT]-3. Dosage dependent suppressor of calmodulin mutants which have specific defects in SPB assembly.
It is an open question how the multiple special and temporal scales involved in intracellular Ca2+ handling within the STDP models affect the plasticity outcomes predicted by these models. Hebbian or associative plasticity is triggered by postsynaptic Ca2+ influx which activates calmodulin and CaMKII. The influx of Ca2+ through voltage-dependent NMDA receptors and Ca2+ channels is regulated by Ca2+ -activated K+ channels (SK-channels) providing negative feedback regulation of postsynaptic [Ca2+]. Using 3-dimensional modelling of Ca2+ and calmodulin dynamics within dendritic spines we show that the non-linear relationship between Ca2+ influx and calmodulin activation endows SK-channels with the ability to "gate" calmodulin activation and therefore the induction of Hebbian synaptic plasticity. Since SK-channels are inhibited by several neuro-modulator receptors including acetylcholine and noradrenaline, the gating of synaptic plasticity by SK-channels could represent a common mechanism by which ...
It is an open question how the multiple special and temporal scales involved in intracellular Ca2+ handling within the STDP models affect the plasticity outcomes predicted by these models. Hebbian or associative plasticity is triggered by postsynaptic Ca2+ influx which activates calmodulin and CaMKII. The influx of Ca2+ through voltage-dependent NMDA receptors and Ca2+ channels is regulated by Ca2+ -activated K+ channels (SK-channels) providing negative feedback regulation of postsynaptic [Ca2+]. Using 3-dimensional modelling of Ca2+ and calmodulin dynamics within dendritic spines we show that the non-linear relationship between Ca2+ influx and calmodulin activation endows SK-channels with the ability to "gate" calmodulin activation and therefore the induction of Hebbian synaptic plasticity. Since SK-channels are inhibited by several neuro-modulator receptors including acetylcholine and noradrenaline, the gating of synaptic plasticity by SK-channels could represent a common mechanism by which ...
Background: Recent genetic studies identified mutations in CALM1 or CALM2, 2 of the 3 human genes encoding calmodulin (CaM), in both catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome (LQTS). CPVT is commonly caused by mutations in sarcoplasmic reticulum genes that increase diastolic Ca leakage through ryanodine receptor (RyR2) Ca relase channels, whereas LQTS is usually caused by dysfunctional plasma membrane ion channels. How mutant CaM causes either CPVT or LQTS is unknown.. Objective: To gain mechanistic insight into how CaM mutations cause divergent human arrhythmia phenotypes.. Methods and Results: We prepared recombinant wild-type (WT) and mutant CaM proteins associated with either CPVT (N54I, N98S) or LQTS ( F142L, D130G). LQTS CaM mutations drastically reduce Ca binding affinity to CaM, whereas CPVT mutations have either no effect (N54I) or slightly reduce Ca binding affinity (N98S). At physiological free CaM [100 nM] and Ca [120 nM], CPVT CaMs ...
In article ,95059.080316RJC8 at psuvm.psu.edu, Richard Cyr ,RJC8 at psuvm.psu.edu, writes: , Does anyone know of a reference wher , e the extinction coefficient of CaM was determined? The Merck index gives the extinction coefficient(1%) at A280 to be 2.1 Christine Dept. of Biochemistry hughe014 at mc.duke.edu ...
(1994) Means. FEBS Letters. Calcium and its ubiquitous intracellular receptor calmodulin are required for cell proliferation. Studies in a variety of model systems are beginning to identify components of the calcium/calmodulin cascade required for movement of quiescent cells into the cell cycle a...
The vacuolar calmodulin (CaM)-stimulated Ca2+-ATPase, BCA1p, in cauliflower (Brassica oleracea) has an extended N terminus, which was suggested to contain a CaM-binding domain (S. Malmstrom, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324328). The goal of the present study was to determine the role of the N terminus in regulating BCA1p. Western analysis using three different antisera showed that the N terminus of BCA1p is cleaved off by trypsin and that the N terminus contains the CaM-binding domain. Furthermore, the expressed N terminus binds CaM in a Ca2+dependent manner. A synthetic peptide corresponding to the CaM-binding domain of BCA1p (Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca2+ pumping by BCA1p in cauliflower low-density membranes, indicating that the CaM-binding region of BCA1p also has an autoinhibitory function. The expressed N terminus of BCA1p and a synthetic peptide (Ala-19 to Met-39) were good substrates for phosphorylation by protein kinase C. Sequencing of the ...
Structural and biophysical studies reveal how CaMKII kinases, which are important for cellular learning and memory, are switched on by binding of Ca2+/calmodulin.
Rabbit polyclonal Calmodulin (phospho T79 + S81) antibody validated for WB, ELISA, IHC, ICC/IF and tested in Human. Referenced in 2 publications. Immunogen…
Adunyah S.E.; Dean W.L., 1987: Regulation of human platelet membrane calcium transport by cyclic amp and calmodulin dependent phosphorylation
Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+regulation of the activity of synthetic smooth-muscle thin filaments Academic Article ...
Many signalling pathways in plants are regulated by the second messenger calcium (Ca2+). In the standard model, Ca2+-sensor proteins, such as CaM (calmodulin), detect Ca2+ signals and subsequently regulate downstream targets to advance the signal transduction cascade. In addition to CaM, plants possess many CMLs (CaM-like proteins) that are predicted to function as Ca2+ sensors, but which remain largely uncharacterized. In the present study, we examined the biochemical properties, subcellular localization and tissue-specific distribution of Arabidopsis CML43. Our data indicate that CML43 displays characteristics typical of Ca2+ sensors, including high-affinity Ca2+ binding, conformational changes upon Ca2+ binding that expose hydrophobic regions and stabilization of structure in the presence of Mg2+ or Ca2+. In vivo localization analysis demonstrates that CML43 resides in cytosolic and nuclear compartments. Transgenic plants expressing a CML43:GUS (β-glucoronidase) promoter reporter gene ...
The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was
The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. ...
Forkhead Transcription Factor; Drives S-phase Specific Expression Of Genes Involved In Chromosome Segregation, Spindle Dynamics, And Budding; Suppressor Of Calmodulin Mutants With Specific SPB Assembly Defects; Telomere Maintenance Role; Regulates Replicative Lifespan; Ortholog Of C. Elegans Lifespan Regulator PHA-4
Protein interactions animation. Clip 10 of 10. Final clip in an animation sequence showing protein interactions within a dividing cell. This clip, which includes labels, shows an actin-myosin bundle (right) forming part of the contractile ring at the periphery of the cell. Proteins (left) are in the surrounding cytoplasm, including those that activate the contractile ring. This contraction (a form of cytokinesis) is initiated by a wave of calcium ions (see K003/3843). Here, the heads of the myosin filaments (dark) are wrapped around the actin filaments (light) and pulling them. The full sequence of ten clips shows the interaction between the protein calmodulin (CaM, calcium-modulated protein), calcium ions, the MLCK (myosin light-chain kinase) protein, and the actin-myosin bundles of the cells cytoskeleton, resulting in contraction of the membrane and cell division. For the entire sequence, see clips K003/3847 to K003/3838. For the same sequence as an anaglyph 3D animation, see clips K003/4492 to K003
Authors: Bertini, Ivano; Luchinat, Claudio; Parigi, Giacomo; Yuan, Jing. Citation: Bertini, Ivano; Kursula, Petri; Luchinat, Claudio; Parigi, Giacomo; Vahokoski, Juha; Wilmanns, Matthias; Yuan, Jing. "Accurate Solution Structures of Proteins from X-ray Data and a Minimal Set of NMR Data: Calmodulin-Peptide Complexes As Examples" J. Am. Chem. Soc. 131, 5134-5144 (2009).. Assembly members: ...
Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via ...
Roles of calmodulin and CaMKII in mediating PKG stimulation of Kir6.2/SUR2A channels.Recombinant Kir6.2/SUR2A channels were expressed in HEK293 cells by transie
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Background CALM2 is the second calmodulin gene. Although the CALM1,CALM2, and CALM3 calmodulin proteins are identical, at the nucleotide level they share only about 80% identity within their coding regions, and they contain...
Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for ...
View mouse Camta1 Chr4:151059525-151861876 with: phenotypes, sequences, polymorphisms, proteins, references, function, expression
The Ca2+-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to Ano1 or whether phosphorylation or additional Ca2+-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca2+ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca2+-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca2+-activated K+ (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba2+, which does not activate CaM. In addition, we conclude that reversible ...
I need an online or downloadable hydropathy plot programme. But I need something special! I want a programme that takes into account the presence or absence of non-covalent modifications, especially phosphorylation sites and calmodulin binding sites. Any suggestions? If so, please email me at: o.s.depeyer at reading.ac.uk ...
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Myc-DDK-tagged ORF clone of Homo sapiens calmodulin-like 4 (CALML4), transcript variant 1 as transfection-ready DNA - 10 µg - OriGene - cdna clones
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TY - JOUR. T1 - Phosphorylation of smooth muscle myosin light chain kinase by Ca2+/calmodulin-dependent protein kinase II. T2 - Comparative study of the phosphorylation sites. AU - Hashimoto, Yoshiaki. AU - Soderling, Thomas. PY - 1990. Y1 - 1990. N2 - Smooth muscle myosin light chain kinase (MLC-kinase) was rapidly phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) to a molar stoichiometry of 2.77 ± 0.15 associated with a threefold increase in the concentration of calmodulin (CaM) required for half-maximal activation of MLC-kinase. Binding of CaM to MLC-kinase markedly reduced the phosphorylation stoichiometry to 0.21 ± 0.05 and almost completely inhibited phosphorylation of sites in two peptides (32P-peptides P1 and P2) with reduced phosphorylation of peptide P3. By analogy, cAMP-dependent protein kinase phosphorylated MLC-kinase to a stoichiometry of 3.0 or greater in the absence of CaM with about a threefold decrease in the ...
TY - JOUR. T1 - Definition of the Inhibitory Domain of Smooth Muscle Myosin Light Chain Kinase by Site-Directed Mutagenesis. AU - Ito, Masaaki. AU - Guerriero, Vince. AU - Chen, Xiaomin. AU - Hartshorne, David J.. PY - 1991/4/1. Y1 - 1991/4/1. N2 - Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. ...
We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin Is. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light ...
The effect of calmodulin on the order of lipids in rhodopsin-free and rhodopsin-containing membranes has been studied using spin-label electron spin resonance methods. Calmodulin, up to 10(-6)M, did not change the measured order of lipids in bilayer membranes containing only rhodopsin. However, for bovine rod outer segment disc membranes, which contain rhodopsin and other proteins, calmodulin induced a significant concentration and temperature dependent increase in the order of the membrane lipids. This suggests that the site of calmodulin binding is remote from rhodopsin itself, and the nature of the binding appears to be a membrane surface phenomenon.
The Munc13 proteins are the key mediators of synaptic vesicle priming, an essential step in Ca2+-regulated neurotransmitter release that renders docked vesicles fusion-competent prior to exocytosis. They have emerged as important regulators of adaptive synaptic mechanisms such as presynaptic short-term plasticity, a process by which the release of neurotransmitter is dynamically adapted to a changing demand. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+-dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. This study aimed to (i) establish whether such regulation through CaM exists in the other Munc13 isoforms, bMunc13-2 and Munc13-3, and (ii) provide structural insights into the Munc13-CaM interaction. Bioinformatic tools were used to identify potential CaM recognition motifs in the non-conserved sequences of bMunc13-2 and Munc13-3. ...
Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone ...
TY - JOUR. T1 - Degradation of PEP-19, a calmodulin-binding protein, by calpain is implicated in neuronal cell death induced by intracellular Ca2+ overload. AU - Kanazawa, Y.. AU - Makino, M.. AU - Morishima, Y.. AU - Yamada, K.. AU - Nabeshima, T.. AU - Shirasaki, Y.. PY - 2008/6/23. Y1 - 2008/6/23. N2 - Excessive elevation of intracellular Ca2+ levels and, subsequently, hyperactivation of Ca2+/calmodulin-dependent processes might play an important role in the pathologic events following cerebral ischemia. PEP-19 is a neuronally expressed polypeptide that acts as an endogenous negative regulator of calmodulin by inhibiting the association of calmodulin with enzymes and other proteins. The aims of the present study were to investigate the effect of PEP-19 overexpression on cell death triggered by Ca2+ overload and how the polypeptide levels are affected by glutamate-induced excitotoxicity and cerebral ischemia. Expression of PEP-19 in HEK293T cells suppressed calmodulin-dependent signaling and ...