WorldWideScience

Sample records for calmodulin

  1. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  2. Role of Calmodulin in Cell Proliferation

    Science.gov (United States)

    Chafouleas, J.

    1983-01-01

    Calmodulin levels were found to increase as cells enter plateau. The data suggest that the cells are exiting the cell cycle late in the G sub 1 phase, or that the calmodulin levels in plateau cells are uncoupled to progression into S phase in plateau cells. Upon release, calmodulin levels rapidly decrease. Following this decrease, there is a increase prior to S phase.

  3. Calmodulin Binding Proteins and Alzheimer's Disease.

    Science.gov (United States)

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  4. Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

    International Nuclear Information System (INIS)

    An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. The epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or toponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase

  5. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Barreda, Elena Gomez de [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Avila, Jesus, E-mail: javila@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); CIBER de Enfermedades Neurodegenerativas, 28031 Madrid (Spain)

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  6. Mechanical stretching of proteins: calmodulin and titin

    Science.gov (United States)

    Cieplak, Marek

    2005-07-01

    Mechanical unfolding of several domains of calmodulin and titin is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. It is shown that this simple model captures the experimentally observed difference between the two proteins: titin is a spring that is tough and strong whereas calmodulin acts like a weak spring with featureless force-displacement curves. The difference is related to the dominance of the α secondary structures in the native structure of calmodulin. The tandem arrangements of calmodulin unwind simultaneously in each domain whereas the domains in titin unravel in a serial fashion. The sequences of contact events during unravelling are correlated with the contact order, i.e., with the separation between contact making amino acids along the backbone in the native state. Temperature is found to affect stretching in a profound way.

  7. Mechanical Stretching of Proteins: Calmodulin and Titin

    OpenAIRE

    Cieplak, Marek

    2004-01-01

    Mechanical unfolding of several domains of calmodulin and titin is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. It is shown that this simple model captures the experimentally observed difference between the two proteins: titin is a spring that is tough and strong whereas calmodulin acts like a weak spring with featureless force-displacement curves. The difference is related to the dominance of the alpha secondary structures in the native s...

  8. Developmental differences in posttranslational calmodulin methylation in pea plants

    International Nuclear Information System (INIS)

    A calmodulin-N-methyltransferase was used to analyze the degree of lysine-115 methylation of pea calmodulin. Calmodulin was isolated from segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by the calmodulin methyltransferase in the presence of 3H-methyl-S-adenosylmethionine. Calmodulin methylation levels were lower in apical root segments and in the young lateral roots compared with the mature, differentiated root tissues. The methylation of these calmodulin samples occurs specifically at lysine 115 since site-directed mutants of calmodulin with substitutions at this position were not methylated and competitively inhibited methylation. The present findings, combined with previous data showing differences in NAD kinase activation by methylated and unmethylated calmodulins, raise the possibility that posttranslational methylation could affect calmodulin action

  9. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    SUN; Daye(

    2001-01-01

    [1]Cheng. W. Y., Cyclic 3', 5'-nucleotide phosphodiestrase: demonstration of an activator, Biochm. Biophys. Res. Commun.,1970, 38: 533-538.[2]Boynton, A. L., Whitfield, J. F., MacManus, J. P., Calmodulin stimulates DNA synthesis by rat liver cells, BBRC.1980,95(2): 745-749.[3]Gorbacherskaya, L. V., Borovkova, T. V., Rybin, U. O. et al., Effect of exogenous calmodulin on lymphocyte proliferation in normal subjects, Bull Exp. Med. Biol., 1983, 95: 361-363.[4]Wong, P. Y.-K., Lee, W. H., Chao, PH.-W., The role of calmodulin in prostaglandin metabolism, Ann. NY Acad. Sci.,1980, 356: 179-189.[5]Mac Neil, S., Dawson, R. A., Crocker, G. et al., Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth, J. Invest. Dermatol., 1984, 83: 15-19.[6]Crocker, D. G., Dawson, R. A., Mac Neil, S. et al., An extracellular role for calmodulin-like activity in cell proliferation,Biochem. J., 1988, 253: 877-884.[7]Polito. V. S., Calmodulin and calmodulin inhibitors: effect on pollen germination and tube growth, in Pollen: Biology and Implications for Plant Breeding (eds. Mulvshy, D. L., Ottaviaro, E.), New York: Elsevier, 1983.53-60.[8]Biro, R. L., Sun, D. Y., Roux, S. J.et al., Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution, Plant Physiol., 1984,75: 382-386.[9]Terry, M. E., Bonner, B. A., An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of IAA-induced growth, Plant Physiol., 1980, 66: 321-325.[10]Josefina, H. N., Aldasars, J. J., Rodriguez, D., Localization of calmodulin on embryonic Cice aricium L, in Molecular and Cellular Aspects of Calcium in Plant Development (ed. Trewavas, A. J.), New York, London: Plenum Press, 1985, 313.[11]Dauwalder, M., Roux, S. J., Hardison, L., Distribution of calmodulin in pea seedling: immunocytochemical localization in plumules and root apices, Planta, 1986, 168: 461

  10. Functional in vitro test of calmodulin antagonism: effect of drugs on interaction between calmodulin and glycolytic enzymes.

    Science.gov (United States)

    Orosz, F; Christova, T Y; Ovádi, J

    1988-06-01

    A simple procedure has been elaborated to screen for the calmodulin antagonist effect of drugs. A covalently attached fluorescent probe was used to monitor the binding of enzymes known as target enzymes to calmodulin. Moreover, the probe made it possible to recognize a new target enzyme, aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), for calmodulin among glycolytic enzymes. The calmodulin antagonist trifluoperazine prevented or eliminated the complex formation between calmodulin and enzymes studied in reconstituted systems; the Ca channel blockers had no effect. The functional consequences of the effect of drugs on calmodulin-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) interaction were investigated as well. Whereas trifluoperazine suspended the calmodulin-mediated hysteretic inactivation of phosphofructokinase, Ca channel blockers (verapamil and nifedipine) were ineffective. Fendiline (regarded as a Ca channel blocker) seems to act as a functional calmodulin antagonist. Its binding to calmodulin does not prevent the complex formation of phosphofructokinase and calmodulin, but within this ternary complex phosphofructokinase preserves or recovers its original activity measured in the absence of calmodulin. The possible molecular effect of drugs on a calmodulin-enzyme complex is discussed. PMID:2837637

  11. Immunoelectron microscopic localization of calmodulin in corn root cells

    Institute of Scientific and Technical Information of China (English)

    LIJIAXU; JIEWENLIU; DAYESUN

    1993-01-01

    Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.

  12. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  13. Calmodulin Binding Proteins and Alzheimer’s Disease

    Science.gov (United States)

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  14. Structural basis for activation of calcineurin by calmodulin

    OpenAIRE

    Rumi-Masante, Julie; Rusinga, Farai I.; Lester, Terrence E.; Dunlap, Tori B.; Williams, Todd D.; Dunker, A. Keith; Weis, David D.; Trevor P Creamer

    2011-01-01

    The highly conserved phosphatase calcineurin plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin. Calmodulin binds to a regulatory domain within calcineurin, causing a conformational change that displaces an autoinhibitory domain from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which calmodulin acti...

  15. Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

    International Nuclear Information System (INIS)

    The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly

  16. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Science.gov (United States)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  17. The Effect of Calcium on the Binding of Calmodulin to Calcium/Calmodulin Protein Kinase II.

    Science.gov (United States)

    Porta, Angela R.

    2000-01-01

    Introduces a follow-up laboratory experiment demonstrating the formation change when calcium binds to calmodulin. This conformation change allows this complex to bind to a target protein. Presents the necessary information to conduct the experiment and discusses the results. (YDS)

  18. Chronic amphetamine treatment increases striatal calmodulin in rats

    International Nuclear Information System (INIS)

    A radioimmunoassay was developed to measure calmodulin in striatum from rats treated with one dose or repeated injections of amphetamine. Chronic, but not acute, amphetamine treatment resulted in a significant increase in total calmodulin levels in striatal homogenates. This effect may be linked to the behavioral sensitization which develops after chronic amphetamine treatments. (Auth.)

  19. Calmodulin binds to and inhibits the activity of phosphoglycerate kinase.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2004-09-17

    Phosphoglycerate kinase (PGK) functions as a cytoplasmic ATP-generating glycolytic enzyme, a nuclear mediator in DNA replication and repair, a stimulator of Sendai virus transcription and an extracellular disulfide reductase in angiogenesis. Probing of a developmental expression library from Dictyostelium discoideum with radiolabelled calmodulin led to the isolation of a cDNA encoding a putative calmodulin-binding protein (DdPGK) with 68% sequence similarity to human PGK. Dictyostelium, rabbit and yeast PGKs bound to calmodulin-agarose in a calcium-dependent manner while DdPGK constructs lacking the calmodulin-binding domain (209KPFLAILGGAKVSDKIKLIE228) failed to bind. The calmodulin-binding domain shows 80% identity between diverse organisms and is situated beside the hinge and within the ATP binding domain adjacent to nine mutations associated with PGK deficiency. Calmodulin addition inhibits yeast PGK activity in vitro while the calmodulin antagonist W-7 abrogates this inhibition. Together, these data suggest that PGK activity may be negatively regulated by calcium and calmodulin signalling in eukaryotic cells. PMID:15363631

  20. Acidic/IQ Motif Regulator of Calmodulin*

    OpenAIRE

    Putkey, John A.; Waxham, M. Neal; Gaertner, Tara R.; Brewer, Kari J.; Goldsmith, Michael; Kubota, Yoshihisa; Kleerekoper, Quinn K.

    2007-01-01

    The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca2+ binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca2+ binding to sites III and IV, and we present a model showing that this could increase Ca2+ binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ ...

  1. Melittin binding causes a large calcium-dependent conformational change in calmodulin.

    OpenAIRE

    Kataoka, M.(LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France); Head, J F; Seaton, B A; Engelman, D M

    1989-01-01

    The interaction between calmodulin and its target protein is a key step in many calcium-regulated cellular functions. Melittin binds tightly to calmodulin in the presence of calcium and is a competitive inhibitor of calmodulin function. Using melittin as a model for the target peptide of calmodulin, we have found a large Ca2+-dependent conformational change of calmodulin in solution induced by peptide binding. Mg2+ does not substitute for Ca2+ in producing the conformation change. Small-angle...

  2. Mediation of flowering by a calmodulin-dependent proteinkinase

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A calmodulin-dependent protein kinase (MCK1) appeared important in regulating flowering in tobacco. The expression of modified MCK1 that lacks the C-terminal including calmodulin-binding domain upsets the flowering developmental program, leading to the abortion of flower primordia initiated on the main axis of the plant and, as well, caused the prolongation of the vegetative phase in axillary buds. The abortion process of flowers began first in the developing anthers and subsequently the entire flower senesces. In axillary buds the prolonged vegetative phase was characterized by atypical elongated, narrow, twisted leaves. These results suggested a role for calmodulin-dependent protein kinase homologs in mediating flowering.

  3. Dopamine binds calmodulin during autoregulation of dopaminergic D2 receptor signaling through CaMKIIα-calmodulin complex.

    Science.gov (United States)

    Laoye, B J; Okurumeh, O A; Obagaye, O V; Olagunju, M O; Bankole, O O; Olubiyi, O O; Ogundele, O M

    2016-06-01

    The role of dopaminergic D2 receptor (D2R) autoregulation in dopamine (DA) neurotransmission cannot be overemphasized in cause and progression of disorders associated with complex behaviors. Although previous studies have shown that D2R is structurally and physiologically linked with calcium/calmodulin-dependent kinase II (CaMKIIα), however, the role of calmodulin in the CaMKIIα complex in D2R regulation remains elusive. In this study, using structural biology modeling softwares (iGEMDOCK and CueMol), we have shown the interaction between D2R, CaMKIIα, calmodulin, and DA under varying conditions. The outcomes of this study suggest that CaMKIIα causes a change in DA binding affinity to the D2R receptive site while the detached DA binds to calmodulin to stop the activity of D2R in the D2R-dopaminergic D1 receptor (D1R) heteromer. Ultimately, we concluded that D2R autoregulates to stop its heteromeric combination with D1R. D2R interacts with D1R to facilitate calcium movement that activates calmodulin, then CaMKIIα. The CaMKIIα-calmodulin complex changes the affinity of DA-D2R causing DA to break free and bind with calmodulin. PMID:26446938

  4. Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

    Institute of Scientific and Technical Information of China (English)

    Zi Fang; Zhenguang Yan; Shuo Li; Qin Wang; Weizhong Cao; Guangrui Xu; Xunhao Xiong; Liping Xie; Rongqing Zhang

    2008-01-01

    Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinct-ada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata.

  5. Unbiased simulation of structural transitions in calmodulin

    CERN Document Server

    Zuckerman, D M

    2003-01-01

    We introduce an approach for performing "very long" computer simulations of the dynamics of simplified, folded proteins. Using an alpha-carbon protein model and a fine grid to mimic continuum computations at increased speed, we perform unbiased simulations which exhibit many large-scale conformational transitions at low cost. In the case of the 72-residue N-terminal domain of calmodulin, the approach yields structural transitions between the calcium-free and calcium-bound structures at a rate of roughly one per day on a single Intel processor. Stable intermediates can be clearly characterized. The model employs Go-like interactions to stabilize two (or more) experimentally-determined structures. The approach is trivially parallelizable and readily generalizes to more complex potentials at minimal cost.

  6. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    Directory of Open Access Journals (Sweden)

    Cyr Janet L

    2002-11-01

    Full Text Available Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3 in its neck region; we identified a fourth IQ domain (IQ4, located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. Conclusions We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

  7. Regulation of brain adenylate cyclase by calmodulin

    International Nuclear Information System (INIS)

    This thesis examined the interaction between the Ca2+-binding protein, calmodulin (CaM), and the cAMP synthesizing enzyme, adenylate cyclase. The regulation of guanyl nucleotide-dependent adenylate cyclase by CaM was examined in a particulate fraction from bovine striatum. CaM stimulated basal adenylate cyclase activity and enhanced the stimulation of the enzyme by GTP and dopamine (DA). The potentiation of GTP- and DA-stimulated adenylate cyclase activities by CaM was more sensitive to the concentration of CaM than was the stimulation of basal activity. A photoreactive CaM derivative was developed in order to probe the interactions between CaM and the adenylate cyclase components of bovine brain. Iodo-[125I]-CaM-diazopyruvamide (125I-CAM-DAP) behaved like native CaM with respect to Ca2+-enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca2+-dependent stimulation of adenylate cyclase. 125I-CaM-DAP cross-linked to CaM-binding proteins in a Ca2+-dependent, concentration-dependent, and CaM-specific manner. Photolysis of 125I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000

  8. Conformational heterogeneity of the calmodulin binding interface

    Science.gov (United States)

    Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.

    2016-04-01

    Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

  9. Calmodulin modulation of ion channels and receptors

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Ion channels and receptors are the structural basis for neural signaling and transmission. Recently, the function of ion channels and receptors has been demonstrated to be modulated by many intracellular and extracellular chemicals and signaling molecules. Increasing evidence indicates that the complexity and plasticity of the function of central nervous system is determined by the modulation of ion channels and receptors. Among various mechanisms, Ca 2+ signaling pathways play important roles in neuronal activity and some pathological changes. Ca 2+ influx through ion channels and receptors can modulate its further influx in a feedback way or modulate other ion channels and receptors. The common feature of the modulation is that Ca 2+ /calmodulin (CaM) is the universal mediator. CaM maintains the coordination among ion channels/receptors and intracellular Ca 2+ homeostasis by feedback modulation of ion channels/receptors activity. This review focuses on the modulating processes of ion channels and receptors mediated by CaM, and further elucidates the mechanisms of Ca 2+ signaling.

  10. Extracellular calmodulin: A polypeptide signal in plants?

    Institute of Scientific and Technical Information of China (English)

    孙大业; 唐文强; 马力耕

    2001-01-01

    Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca2+ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation,and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore,we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein,phospholipase C, IP3, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.

  11. The chemosensitizing agent lubeluzole binds calmodulin and inhibits Ca(2+)/calmodulin-dependent kinase II.

    Science.gov (United States)

    Bruno, Claudio; Cavalluzzi, Maria Maddalena; Rusciano, Maria Rosaria; Lovece, Angelo; Carrieri, Antonio; Pracella, Riccardo; Giannuzzi, Giulia; Polimeno, Lorenzo; Viale, Maurizio; Illario, Maddalena; Franchini, Carlo; Lentini, Giovanni

    2016-06-30

    An affinity capillary electrophoresis (ACE) method to estimate apparent dissociation constants between bovine brain calmodulin (CaM) and non-peptidic ligands was developed. The method was validated reproducing the dissociation constants of a number of well-known CaM ligands. In particular, the potent antagonist 125-C9 was ad hoc synthesized through an improved synthetic procedure. The ACE method was successfully applied to verify CaM affinity for lubeluzole, a well-known neuroprotective agent recently proved useful to potentiate the activity of anti-cancer drugs. Lubeluzole was slightly less potent than 125-C9 (Kd = 2.9 ± 0.7 and 0.47 ± 0.06 μM, respectively) and displayed Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibition (IC50 = 40 ± 1 μM). Possible binding modes of lubeluzole to CaM were explored by docking studies based on the X-ray crystal structures of several trifluoperazine-CaM complexes. An estimated dissociation constant in good agreement with the experimental one was found and the main aminoacidic residues and interactions contributing to complex formation were highlighted. The possibility that interference with Ca(2+) pathways may contribute to the previously observed chemosensitizing effects of lubeluzole on human ovarian adenocarcinoma and lung carcinoma cells are discussed. PMID:27043269

  12. Mediation of flowering by a calmodulin-dependent proteinkinase

    Institute of Scientific and Technical Information of China (English)

    LIANG; Shuping(

    2001-01-01

    [1]Roberts. D. M., Harmon, A. C., Calcium-modulated proteins: Targets of the intracellular signals in higher plants, Ann. Rev.Plant Physiol. Plant Mol. Biol., 1992, 43: 375-414.[2]Sun. D. Y.. Bian, Y. Q., Zhao, B. H. et al., The effects of extracellular calmodulin on cell wall regeneration of protoplasts and cell division, Plant Cell Physiol., 1995, 36: 133-138.[3]Hrabak, E M., Dickmann, L. J., Satterlee, J. S. et al., Characterization of eight new members of the calmodulin-like domain protein kinase gene family from A rabidopsis thaliana, Plant Mol. Biol., 1996, 31:405-412.[4]Huang, J. F., Teyton, L., Harper, J, F., Activation of a Ca2+-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain, Biochemistry, 1996, 35: 13222-13234.[5]Yoo, B. C., Harmon, A. C., Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain, Biochem., 1996, 35: 12029-12037.[6]Saijo, Y., Hata, S., Sheen, J. et al., cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases,Biochem. Biophys. Acta, 1997, 1350: 109-114.[7]Neuhaus. G., Bowler, C., Kern, R. et al., Calcium/calmodulin-dependent and -independent phytochrome signal transduction pathways, Cell, 1993, 73: 937-952.[8]Yang, T., Poovaiah, B. W., Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action, J. Biol. Chem., 2000, 275(5): 3137-3143.[9]Watillon, B., Kettmenn, R., Boxus, P. et al., Calcium/calmodulin-binding serine/threonine protein kinase homologous to mammalian type II calcium/calmodulin-dependent protein kinase is expressed in plant cells, Plant Physiol., 1993, 101:1381-1384.[10]Baum, G., Lev-Yadun, S., Fridmann, Y. et al., Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants, EMBO J, 1996, 15: 2988-2996.[11]Lu, Y. T., Dharmasiri, M. A. N., Harrington

  13. Studies on a novel macrophage-specific calmodulin binding glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Orlow, S.J.

    1986-01-01

    The murine macrophage-like cell line J774 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin-binding protein which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and these elicited with concanavalin A, lipopolysaccharide, proteose peptone or Bacillus Clamette Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed using a rabbit antiserum raised to the partially purified calmodulin binding protein and (/sup 125/I) calmodulin covalently crosslinked to the principal calmodulin binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of molecular weight 50,000 to 60,000 was isolated. It could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property plus its sensitivity to endoglycosidase F led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization and evidence of glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for calcium-calmodulin.

  14. Studies on a novel macrophage-specific calmodulin binding glycoprotein

    International Nuclear Information System (INIS)

    The murine macrophage-like cell line J774 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin-binding protein which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and these elicited with concanavalin A, lipopolysaccharide, proteose peptone or Bacillus Clamette Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed using a rabbit antiserum raised to the partially purified calmodulin binding protein and (125I) calmodulin covalently crosslinked to the principal calmodulin binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of molecular weight 50,000 to 60,000 was isolated. It could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property plus its sensitivity to endoglycosidase F led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization and evidence of glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for calcium-calmodulin

  15. Mutations in calmodulin cause ventricular tachycardia and sudden cardiac death

    DEFF Research Database (Denmark)

    Nyegaard, Mette; Overgaard, Michael Toft; Sondergaard, M.T.; Vranas, Marta; Behr, Elijah R.; Hildebrandt, L.L.; Lund, J.; Hedley, Paula L.; Camm, A. John; Wettrell, Göran; Fosdal, Inger; Christiansen, Michael; Borglum, Anders D.

    2012-01-01

    substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe......Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a...... calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac...

  16. Identification of calmodulin released by osmotic shock of maize roots

    International Nuclear Information System (INIS)

    Exogenously applied calcium at low concentrations (10 mM and less) stimulates, while higher concentrations (greater than 20 mM) inhibit maize root growth. The phenothiazine calmodulin inhibitors chlorpromazine and trifluoperzine inhibit maize root growth and are reversible by calcium. The loss of acid-inducible growth after osmotic shock indicates that at least part of the complex associated the acid-induced growth is released. Since calmodulin (CaM) is a small protein (mol wt about 17 kD) found to play a pivotal role in Ca+2 regulated mechanisms, the material released from maize roots by osmotic shock was examined for the presence of CaM

  17. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    International Nuclear Information System (INIS)

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca2+. One mechanism by which Ca2+ may trigger neutrophil activation is through Ca2+/calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca2+/CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and 125I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound 125I-CaM in a Ca2+-dependent manner. One predominant region of 125I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca2+-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 μg/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca2+-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca2+/calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase

  18. Bending of the calmodulin central helix : A theoretical study

    NARCIS (Netherlands)

    VanderSpoel, D; DeGroot, BL; Hayward, S; Berendsen, HJC; Vogel, HJ

    1996-01-01

    The crystal structure of calcium-calmodulin (CaM) reveals a protein with a typical dumbbell structure. Various spectroscopic studies have suggested that the central linker region of CaM, which is alpha-helical in the crystal structure, is flexible in solution. In particular, NMR studies have indicat

  19. Intracellular levels of calmodulin are increased in transformed cells

    Institute of Scientific and Technical Information of China (English)

    WANG; HONGQINGZHANG; 等

    1992-01-01

    By using Hoechst 33342,rabbit anti calmodulin antibody,FITC-labeled goat anti rabbit IgG and SR101(sulfo rhodamine 101)simultaneously to stain individual normal and transformed cells,the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus,calmodulin and total protein respectively,could be recognized in individualj cells without interference,The phase of the cell cycle was determined by DNA content(Hoechst 33342),We found that in transformed cells(NIH3T3) tsRSV-LA90,cultured at 33℃ and transformed C3H10T1/2 Cells),the ration of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV-LA90 cells,cultured at 39℃ and C3H10T1/2 cells)in every cell cycle phase,This ration increased obviously only from G1 to S phase in either normal or transformed cells.The results showed that calmodulinreally increased during the transformation,and its increase was specific.In the meantime when cells proceeded from G1 to S.the intraceollular calmodulin content also increased specifically.

  20. Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes

    DEFF Research Database (Denmark)

    Ye, Q; Wei, Y; Fischer, R;

    1997-01-01

    Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for protein...

  1. Analysis of the state of posttranslational calmodulin methylation in developing pea plants

    International Nuclear Information System (INIS)

    A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [3H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity

  2. Fluorescence Spectra Studies on the Interaction between Lanthanides and Calmodulin

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    The conformation of Calmodulin(CaM) induced by lanthanides has been examined using fluorescence methods.With the addition of lanthanide (Ln3+), the intrinsic fluorescence intensity of CaM without calcium ions (Apo-CaM) first increases and then decreases.Ln3+ causes the decrease of intrinsic fluorescence intensity of calcium saturated CaM (Ca2+4-CaM) only at high concentrations.At low concentrations, Ln3+ results not only in the enhancement of fluorescence intensity of Apo-CaM, but also in a blue shift of the maximum emission wavelengh of dansyl labeled calmodulin(Apo-D-CaM).The molecular mechanism of the interaction between Ln3+ and CaM has been discussed in the light of the fluorescence spectra.

  3. Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens.

    OpenAIRE

    Nakazawa, K; Higo, K.; Abe, K; Tanaka, Y.; Saito, H; Matsuki, N.

    1993-01-01

    1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced sp...

  4. Calmodulin and calmodulin-binding proteins in cystic fibrosis and normal human fibroblasts

    International Nuclear Information System (INIS)

    The authors have investigated the possibility that a lesion in a calmodulin (CaM)-dependent regulatory mechanism may be involved in cystic fibrosis (CF). The level of CaM, CaM-binding proteins (CaM-BP) and a CaM-dependent phosphatase (CaM-Ptase) have been compared in cultured fibroblasts from CF patients versus age- and sex-matched control subjects. The CaM concentration, measured by radioimmunoassay, ranged from 0.20 to 0.76 μg/mg protein (n=8); there was no significant difference in the average CaM concentration from CF patients vs controls. Using Western blotting techniques with 125I-CaM, they detected at least ten distinct CaM-BPs in fibroblasts with molecular weights ranging from 230K to 37K; the only consistent difference between control and CF cell lines was in a 46.5K CaM-BP, which was depressed in all three CF samples. The 46.5 K CaM-BP was found only in the particulate fraction. A 59K CaM-BP was identified as a CaM-Ptase by its crossreactivity with an antibody against a brain CaM-Ptase. There was no significant difference in CaM-Ptase activity or in the amount of the phosphatase as determined by radioimmunoassay in CF vs. normal samples (n=8). Thus, the level of CaM as well as its various enzymes and proteins do not appear to be altered in CF fibroblasts except for a CaM-BP of 46.5K, the identity of which is currently being investigated

  5. Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

    Science.gov (United States)

    Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

    1996-01-01

    A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

  6. A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

    Science.gov (United States)

    Harmat, V; Böcskei, Z; Náray-Szabó, G; Bata, I; Csutor, A S; Hermecz, I; Arányi, P; Szabó, B; Liliom, K; Vértessy, B G; Ovádi, J

    2000-03-31

    An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin. PMID:10731425

  7. Calmodulin gene expression in response to mechanical wounding and Botrytis cinerea infection in tomato fruit

    Science.gov (United States)

    Calmodulin, a ubiquitous calcium sensor, plays an important role in decoding the stress-triggered intracellular calcium changes and regulates the functions of numerous target proteins involved in various physiological responses in plants. To determine the functional significance of calmodulin in fl...

  8. Facilitation of plateau potentials in turtle motoneurones by a pathway dependent on calcium and calmodulin

    DEFF Research Database (Denmark)

    Perrier, J F; Mejia-Gervacio, S; Hounsgaard, J

    2000-01-01

    1. The involvement of intracellular calcium and calmodulin in the modulation of plateau potentials in motoneurones was investigated using intracellular recordings from a spinal cord slice preparation. 2. Chelation of intracellular calcium with BAPTA-AM or inactivation of calmodulin with W-7 or tr...

  9. Modulation of phosphofructokinase action by macromolecular interactions. Quantitative analysis of the phosphofructokinase-aldolase-calmodulin system.

    Science.gov (United States)

    Orosz, F; Christova, T Y; Ovádi, J

    1988-11-23

    The simultaneous effect of calmodulin and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) on the concentration-dependent behaviour of muscle phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been analysed by means of a covalently attached fluorescent probe, gel penetration experiments, and using a kinetic approach. We found that calmodulin-induced inactivation of phosphofructokinase is suspended by addition of an equimolar amount of aldolase. This effect was attributed to an apparent competition of calmodulin and aldolase for the dimeric forms of kinase. Moreover, the direct binding of aldolase to calmodulin has also been demonstrated, which resulted in a significant decrease in the kcat value of the enzyme. The quantitative analysis of these interactions in the system phosphofructokinase-calmodulin-aldolase is presented. A possible molecular model for the modulation of phosphofructokinase action by macromolecular interactions is envisaged. PMID:2973356

  10. Characterization of a calmodulin binding protein kinase from Arabidopsis thalian

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A full-length calmodulin binding protein kinase cDNA, AtCBK1, from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE. Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests that AtCBK1 encodes a CaM-binding serine/threonine protein kinase.

  11. Identification of spectrin as a calmodulin-binding component in the pituitary gonadotrope

    International Nuclear Information System (INIS)

    Gonadotropin releasing hormone (GnRH) is a hypothalamic decapeptide which stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary. Ca2+ fulfills the requirements of a second messenger for this system. Inhibition of calmodulin will inhibit GnRH stimulated LH release. The aim of the present studies has been to identify the locus of action of calmodulin within the pituitary. By use of an 125I-calmodulin gel overlayer assay, five major Ca2+-dependent 125I-calmodulin labelled components of subunit Mr > 205,000; 200,000; 135,000; 60,000; and 52,000 have been identified. This labeling was found to be phenothiazine-sensitive. Ca2+-independent binding that was observed appears to be due to hydrophobic interactions of calmodulin with acid-soluble proteins, principally histones. Subcellular fractionation revealed that the Ca2+-dependent calmodulin-binding components are localized primarily in the cytosolic fraction. Separation of dispersed anterior pituitary cells through a linear Metrizamide gradient yielded gonadotrope-enriched fractions, which were found to contain all five 125I-calmodulin binding components corresponding to the major bands in the pituitary homogenate. The calmodulin-binding component levels do not appear to be differentially regulated by steroids. The calmodulin binding component with a Mr > 205,000 has been identified as spectrin. Spectrin-like immunoreactivity and 125I-calmodulin-binding activity in pituitary tissue homogenates co-migrated in various percentage acrylamide gels with avian erythrocyte spectrin. Spectrin was detected in a gonadotrope-enriched fraction by immunoblotting, and confirmed in gonadotropes by indirect immunofluorescence of cultured pituitary cells in which spectrin- and LH-immunoreactivity co-localized

  12. Tracking and localization of calmodulin in live cells.

    Science.gov (United States)

    Johnson, Carey K; Harms, Gregory S

    2016-08-01

    The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from 10μm(2)s(-1). For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells. PMID:27113857

  13. Heparin blocks /sup 125/I-calmodulin internalization by isolated rat renal brush border membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meezan, E.; Elgavish, A.; Roden, L.; Wallace, R.W.

    1986-03-05

    /sup 125/I-Calmodulin is internalized by isolated rat renal brush border membrane vesicles (BBV) in a time, temperature and calcium dependent manner. Internalization of /sup 125/I-calmodulin into the osmotically sensitive space of BBV was distinguished from binding of the ligand to the outer BBV surface by examining the interaction of ligand and BBV at different medium osmolarities (300-1100 mosm), uptake was inversely proportional to medium osmolarity. Internalized /sup 125/I-calmodulin was intact and Western blots of solubilized BBV with /sup 125/I-calmodulin demonstrated the presence of several calmodulin-binding proteins of 143, 118, 50, 47.5, 46.5 and 35 kilodaltons which could represent potential intravesicular binding sites for the ligand. Heparin and the related glycosaminoglycan heparin sulfate both showed a dose-dependent inhibition (0.5-50 ..mu..g/ml) of /sup 125/I-calmodulin uptake by BBV, but other sulfated and nonsulfated glycosaminoglycans including chondroitin sulfates, keratan sulfate and hyaluronic acid showed little or no inhibitory effect. Desulfation of heparin virtually abolished the inhibition of uptake while depolymerization reduced it. Heparin did not block the binding of /sup 125/I-calmodulin to BBV proteins as assessed by Western blotting technique suggesting its effect was on internalization of the ligand rather than on its association with internal membrane proteins.

  14. Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

    Science.gov (United States)

    Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

    2000-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

  15. Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: crystallographic and spectroscopic studies.

    Science.gov (United States)

    Vertessy, B G; Harmat, V; Böcskei, Z; Náray-Szabó, G; Orosz, F; Ovádi, J

    1998-11-01

    The modulatory action of Ca2+-calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 A resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3"-(beta-chloroethyl)-2",4"-dioxo-3, 5"-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955-962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions

  16. Barium can replace calcium in calmodulin-dependent contractions of skinned renal arteries of the rabbit.

    Science.gov (United States)

    Kreye, V A; Hofmann, F; Mühleisen, M

    1986-03-01

    Renal arteries of the rabbit were chemically skinned using Triton X-100. In EGTA-buffered solutions containing calmodulin and ATP, small strips of the skinned preparations were found to develop contractile force which was dependent on the concentrations of either free calcium or of free barium. However, a 220 times greater concentration of barium than of calcium was necessary for comparable effects. Quantitatively, the response to barium was dependent on the concentration of calmodulin added to the test solutions. The contractile effect of barium was partly antagonized by the calmodulin antagonist, trifluoperazine. PMID:3960707

  17. Localization of calmodulin in epidermis and skin glands: a comparative immunohistological investigation in different vertebrate species.

    Science.gov (United States)

    Wollina, U; Wevers, A; Mahrle, G

    1991-01-01

    The study deals with the immunolocalization of calmodulin-reactive epithelial cells in different vertebrates (Tinca tinca, Ambystoma mexicanum, Xenopus laevis, Rana ridibunda, Columba domestica, Sus scrofa domestica, Homo sapiens sapiens). The immunoperoxidase technique was performed on acetone fixed frozen sections using monoclonal (BF8) and polyclonal (ACAM) anti-calmodulin antibodies. We were able to differentiate 2 major types of staining patterns: 1. A more superficial epidermal staining in species adapted to an aqueous environment and 2. a staining along the epidermal-dermal junction in species adapted to a terrestrial environment. It seems most likely that epithelial cells immunoreactive for calmodulin are involved in skin permeability control. PMID:1718122

  18. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    OpenAIRE

    Cyr Janet L; Gillespie Peter G

    2002-01-01

    Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much...

  19. Physico-chemical pathways in radioprotective action of calmodulin antagonists

    International Nuclear Information System (INIS)

    Ghost membranes prepared from erythrocytes of Swiss albino mice were irradiated with gamma rays at a dose rate of 0.9 Gy/s. The fluidity of membrane decreased with radiation dose and in the presence of calmodulin antagonists (CA) like chlorpromazine (CPZ), promethazine (PMZ), and trimeprazone (TMZ) it increased. Radiation induced release of Ca2+ from membranes. This release was inhibited by CA mainly by CPZ and PMZ. Being Ca2+ dependent, the changes in the activity of acetylcholine estrase (AchE) following irradiation was also studied. Radiation decreased the activity of AchE in dose dependent manner. Presence of CPZ and PMZ diminished the radiation induced inhibition of AchE but not in the presence of TMZ at the lower concentration tested. It is suggested that apart from scavenging of free radicals, CA perhaps exert their euxoic radioprotective effect through Ca2+ dependent processes. (author)

  20. Calmodulin affects sensitization of Drosophila melanogaster odorant receptors

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    Latha eMukunda

    2016-02-01

    Full Text Available Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs, occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX and a highly conserved co-receptor protein (Orco. The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs expressing Or22a inside the fly’s antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native

  1. A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain

    Science.gov (United States)

    Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.

    1996-01-01

    Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

  2. AKAP150-mediated TRPV1 sensitization is disrupted by calcium/calmodulin

    Directory of Open Access Journals (Sweden)

    Shapiro Mark S

    2011-05-01

    Full Text Available Abstract Background The transient receptor potential vanilloid type1 (TRPV1 is expressed in nociceptive sensory neurons and is sensitive to phosphorylation. A-Kinase Anchoring Protein 79/150 (AKAP150 mediates phosphorylation of TRPV1 by Protein Kinases A and C, modulating channel activity. However, few studies have focused on the regulatory mechanisms that control AKAP150 association with TRPV1. In the present study, we identify a role for calcium/calmodulin in controlling AKAP150 association with, and sensitization of, TRPV1. Results In trigeminal neurons, intracellular accumulation of calcium reduced AKAP150 association with TRPV1 in a manner sensitive to calmodulin antagonism. This was also observed in transfected Chinese hamster ovary (CHO cells, providing a model for conducting molecular analysis of the association. In CHO cells, the deletion of the C-terminal calmodulin-binding site of TRPV1 resulted in greater association with AKAP150, and increased channel activity. Furthermore, the co-expression of wild-type calmodulin in CHOs significantly reduced TRPV1 association with AKAP150, as evidenced by total internal reflective fluorescence-fluorescence resonance energy transfer (TIRF-FRET analysis and electrophysiology. Finally, dominant-negative calmodulin co-expression increased TRPV1 association with AKAP150 and increased basal and PKA-sensitized channel activity. Conclusions the results from these studies indicate that calcium/calmodulin interferes with the association of AKAP150 with TRPV1, potentially extending resensitization of the channel.

  3. Calmodulin Gene Expression in Response to Mechanical Wounding and Botrytis cinerea Infection in Tomato Fruit

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2014-08-01

    Full Text Available Calmodulin, a ubiquitous calcium sensor, plays an important role in decoding stress-triggered intracellular calcium changes and regulates the functions of numerous target proteins involved in various plant physiological responses. To determine the functions of calmodulin in fleshy fruit, expression studies were performed on a family of six calmodulin genes (SlCaMs in mature-green stage tomato fruit in response to mechanical injury and Botrytis cinerea infection. Both wounding and pathogen inoculation triggered expression of all those genes, with SlCaM2 being the most responsive one to both treatments. Furthermore, all calmodulin genes were upregulated by salicylic acid and methyl jasmonate, two signaling molecules involved in plant immunity. In addition to SlCaM2, SlCaM1 was highly responsive to salicylic acid and methyl jasmonate. However, SlCaM2 exhibited a more rapid and stronger response than SlCaM1. Overexpression of SlCaM2 in tomato fruit enhanced resistance to Botrytis-induced decay, whereas reducing its expression resulted in increased lesion development. These results indicate that calmodulin is a positive regulator of plant defense in fruit by activating defense pathways including salicylate- and jasmonate-signaling pathways, and SlCaM2 is the major calmodulin gene responsible for this event.

  4. Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin.

    Directory of Open Access Journals (Sweden)

    Gabriel Gaidos

    Full Text Available Overexpression of the cellular FLICE-like inhibitory protein (cFLIP has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1 of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

  5. The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

    OpenAIRE

    Huang, Hao; Ishida, Hiroaki; Vogel, Hans J.

    2010-01-01

    Soybean calmodulin isoform 4 (sCaM4) is a plant calcium-binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo-) form of sCaM4 possesses a half unfolded structure, with the N-terminal domain unfolded and the C-terminal domain folded. This result was unexpected as the apo-forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high...

  6. Characterization and functional analysis of the calmodulin-binding domain of Rac1 GTPase.

    Directory of Open Access Journals (Sweden)

    Bing Xu

    Full Text Available Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151-164 in Rac1 is essential for calmodulin binding. Within the 151-164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A, activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.

  7. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    Science.gov (United States)

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  8. Impact of methionine oxidation on calmodulin structural dynamics

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin [Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455 (United States); Moen, Rebecca J. [Chemistry and Geology Department, Minnesota State University, Mankato, MN 56001 (United States); Olenek, Michael J. [Biology Department, University of Wisconsin, La Crosse, WI 54601 (United States); Klein, Jennifer C., E-mail: jklein@uwlax.edu [Biology Department, University of Wisconsin, La Crosse, WI 54601 (United States); Thomas, David D., E-mail: ddt@umn.edu [Biochemistry, Molecular Biology and Biophysics Department, University of Minnesota, Minneapolis, MN 55455 (United States)

    2015-01-09

    Highlights: • We measured the distance distribution between two spin labels on calmodulin by DEER. • Two structural states, open and closed, were resolved at both low and high Ca. • Ca shifted the equilibrium toward the open state by a factor of 13. • Methionine oxidation, simulated by glutamine substitution, decreased the Ca effect. • These results have important implications for aging in muscle and other tissues. - Abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron–electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4 nm (closed) and another at ∼6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each

  9. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    O' Day, Danton H., E-mail: danton.oday@utoronto.ca [Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M5S 3G5 (Canada); Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario, Canada L5L 1C6 (Canada); Huber, Robert J. [Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M5S 3G5 (Canada); Suarez, Andres [Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario, Canada L5L 1C6 (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Extracellular calmodulin is present throughout growth and development in Dictyostelium. Black-Right-Pointing-Pointer Extracellular calmodulin localizes within the ECM during development. Black-Right-Pointing-Pointer Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. Black-Right-Pointing-Pointer Extracellular calmodulin exists in eukaryotic microbes. Black-Right-Pointing-Pointer Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca{sup 2+}/CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  10. Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response calmodulin-binding transcription factor AtSR1/CAMTA3

    Science.gov (United States)

    Calcium/calmodulin (Ca2+/CaM) has long been considered a crucial component in wound signaling pathway. However, no functional significance of Ca2+/CaM-binding proteins has been identified in plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca2+/CaM-bindi...

  11. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  12. Structural basis for activation of calcineurin by calmodulin.

    Science.gov (United States)

    Rumi-Masante, Julie; Rusinga, Farai I; Lester, Terrence E; Dunlap, Tori B; Williams, Todd D; Dunker, A Keith; Weis, David D; Creamer, Trevor P

    2012-01-13

    The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein. PMID:22100452

  13. Calmodulin stimulation of calcium transport in carrot microsomal vesicles

    International Nuclear Information System (INIS)

    ATP-dependent 45Ca2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca2+. These results are consistent with the ER being an important site of intracellular Ca2+ regulation

  14. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  15. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  16. Human platelet calmodulin-binding proteins: identification and Ca/sup 2 +/-dependent proteolysis upon platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1987-05-19

    Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and /sup 125/I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound /sup 125/I-calmodulin in a Ca/sup 2 +/-dependent manner; the binding was blocked by ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca/sup 2 +/ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca/sup 2 +/-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca/sup 2 +/ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca/sup 2 +/. The data indicate that limited proteolysis of Ca/sup 2 +//calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca/sup 2 +/ associated with platelet aggregation.

  17. Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607.

    Science.gov (United States)

    Sharma, S; Giri, S; Khuller, G K

    1998-06-01

    A soluble Ca2+/calmodulin dependent protein kinase has been partially purified (approximately 400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC50 of 1.5 and 0.25 microm respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca2+/calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism. PMID:9655195

  18. Effects of calmodulin and calmodulin inhibitors on Ca uptake by sarcoplasmic reticulum of saponin skinned caudal artery

    International Nuclear Information System (INIS)

    Calmodulin (CaM) stimulates plasma membrane transport in many cell types, however, its role in Ca regulation by the sarcoplasmic reticulum (SR) in smooth muscle has not been established. 45Ca uptake was studied in saponin skinned strips of rat caudal artery as a function of CaM and the CaM inhibitors, W-7, calmidazolium (CaMZ), and trifluoperazine (TFP). Although caudal artery strips lose approximately 30% of total tissue CaM during skinning, 0.3 - 2 μM CaM did not increase 45Ca uptake over a wide range of free Ca concentrations (10-8 - 10-6M). Neither W-7 nor CaMZ at concentration of 10-4 - 2 x 10-4M inhibited the MgATP-dependent Ca uptake. Ca uptake was not affected by 50 μM TFP but a significant inhibition was produced by 500 μM. Studies of the effects of TFP on 45Ca efflux indicated that TFP concentrations which inhibited Ca uptake also significantly increased the rate of Ca release. The results suggest that total Ca uptake in caudal artery depends mainly upon MgATP and is not modulated by exogenous CaM or affected by these CaM inhibitors. They cannot preclude that CaM may affect initial velocities or that the CaM inhibitors failed to reach active sites

  19. Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase in free and calmodulin-bound forms

    International Nuclear Information System (INIS)

    A rabbit lung Ca2+-stimulated cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography in the presence of EGTA on DEAE-cellulose and G-200 Sephadex columns still responded to Ca2+ and contained calmodulin (CaM) suggesting that the enzyme exists as a stable CaM-PDE complex. A similar enzyme was demonstrated to exist in bovine lung extract. A monoclonal antibody Cl previously shown to react with the 60 kDa subunit of bovine brain PDE isozymes cross-reacted with the lung enzyme. Purification of the lung enzyme by Cl antibody immunoaffinity chromatography rendered the enzyme dependent of exogenously added CaM for Ca2+ stimulation. The enzyme was further purified by CaM affinity chromatography to near homogeneity. The purified enzyme could be reconstituted into PDE-CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. When reconstitution was carried out in the presence of 45Ca2+, followed by isolation of the protein complex, no 45Ca2+ was found to be associated with the complex. CaM antagonists: trifluoroperazine, compound 48/80 and calcineurin at concentrations abolishing CaM-stimulation of bovine brain PDE had little effect on the bovine lung PDE-CaM complex

  20. Effects of calmodulin and calmodulin inhibitors on Ca uptake by sarcoplasmic reticulum of saponin skinned caudal artery

    Energy Technology Data Exchange (ETDEWEB)

    Stout, M.A.; Silver, P.J.

    1986-03-05

    Calmodulin (CaM) stimulates plasma membrane transport in many cell types, however, its role in Ca regulation by the sarcoplasmic reticulum (SR) in smooth muscle has not been established. /sup 45/Ca uptake was studied in saponin skinned strips of rat caudal artery as a function of CaM and the CaM inhibitors, W-7, calmidazolium (CaMZ), and trifluoperazine (TFP). Although caudal artery strips lose approximately 30% of total tissue CaM during skinning, 0.3 - 2 ..mu..M CaM did not increase /sup 45/Ca uptake over a wide range of free Ca concentrations (10/sup -8/ - 10/sup -6/M). Neither W-7 nor CaMZ at concentration of 10/sup -4/ - 2 x 10/sup -4/M inhibited the MgATP-dependent Ca uptake. Ca uptake was not affected by 50 ..mu..M TFP but a significant inhibition was produced by 500 ..mu..M. Studies of the effects of TFP on /sup 45/Ca efflux indicated that TFP concentrations which inhibited Ca uptake also significantly increased the rate of Ca release. The results suggest that total Ca uptake in caudal artery depends mainly upon MgATP and is not modulated by exogenous CaM or affected by these CaM inhibitors. They cannot preclude that CaM may affect initial velocities or that the CaM inhibitors failed to reach active sites.

  1. Influence of phosphorylation of lymphocyte's plasma-membrane proteins and calmodulin on Ca2+, Mg2+ -ATPase activity under irradiation

    International Nuclear Information System (INIS)

    We establish that the regulation of Ca2+, Mg2+ - ATPase from plasma membranes of rat spleen lymphocytes is controlled by calmodulin and the Ca2+, calmodulin-dependent phosphorylation system. The mechanisms of regulation of this process are sensitive to the total X-ray irradiation in doses of 0.5 and 1 Gy

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

    International Nuclear Information System (INIS)

    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

  3. Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants.

    Science.gov (United States)

    Montgomery, Heather J; Bartlett, Ryan; Perdicakis, Basil; Jervis, Eric; Squier, Thomas C; Guillemette, J Guy

    2003-07-01

    Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite. PMID:12820885

  4. Matricellular signal transduction involving calmodulin in the social amoebozoan dictyostelium.

    Science.gov (United States)

    O'Day, Danton H; Huber, Robert J

    2013-01-01

    The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa) releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes. PMID:24705101

  5. Matricellular Signal Transduction Involving Calmodulin in the Social Amoebozoan Dictyostelium

    Directory of Open Access Journals (Sweden)

    Danton H. O'Day

    2013-02-01

    Full Text Available The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL repeat-containing, calmodulin (CaM-binding protein (CaMBP CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes.

  6. Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Siems, W. F.; Jones, J. P.; Poovaiah, B. W.

    2001-01-01

    The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

  7. Ca2+ binding sites in calmodulin and troponin C alter interhelical angle movements.

    Science.gov (United States)

    Goto, Kunihiko; Toyama, Akira; Takeuchi, Hideo; Takayama, Kazuyoshi; Saito, Tsutomu; Iwamoto, Masatoshi; Yeh, Jay Z; Narahashi, Toshio

    2004-03-12

    Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism. PMID:15013750

  8. Expression of flounder Paralichthys olivaceus calmodulin gene in prokaryotic and eukaryotic cells

    OpenAIRE

    LÜ Ai-Jun; HU Xiu-Cai

    2008-01-01

    The primer was designed based on the sequence which was obtained from subtractive cDNA library, and then cloned the flounder Paralichthys olivaceus calmodulin gene(PoCaM) using SMART cDNA as the model. Computer-assisted analysis revealed the potential open reading frame encoded a protein of 149 amino acids with a predicted size of 17 kDa, the predicted theoretical isoelectric points (PI) is 3.93. Sequence alignment of PoCaM with other well known calmodulin proteins showed a significant homolo...

  9. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.

    Science.gov (United States)

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G; Módos, Károly; Kardos, József; Liliom, Károly

    2010-10-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well. PMID:20522785

  10. Real—time Analysis of the Interaction between Calmodulin and Melittin by SPR Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    WeiGuoLI; XiaoQiangCUI; 等

    2002-01-01

    The dynamic interaction process of calmodulin with an immobilized peptide melittin was investigated in real time by surface plasmon resonance spectroscopy, and dissociation constant of the complex was calculated to be 3.37×10-6 mol/L.

  11. Effect of calmodulin antagonists on contraction and45Ca movements in rat aorta

    NARCIS (Netherlands)

    Wermelskirchen, D.; Koch, P.; Wilhelm, D.; Nebel, U.; Leidig, A.; Wilffert, B.; Peters, Thies

    1989-01-01

    To study the selectivity of calmodulin antagonists it was assumed that they should inhibit noradrenaline (NA)- and K+-induced contractions similarly without an accompanying inhibition of45Ca uptake. Therefore, in isolated rat aorta the effects of W-7, calmidazolium and trifluoperazine on contraction

  12. Role of calmodulin (δ-subunit) in activation of phosphorylase kinase from rabbit skeletal muscles

    International Nuclear Information System (INIS)

    The structure of the inactivated and activated forms of phospholyase kinase was compared. The enzyme was activated by incubation in an alkaline medium (pH 8.5), phosphorylation of the catalytic subunit of cAMP-dependent protein kinase, and limited proteolysis. Hydrophobic chromatography on phenyl-Sepharose and electrophoresis in a polyacrylamide gel density gradient were employed for a comparison of these forms of the enzyme. Activation of the enzyme was accompanied by the separation of a low-molecular-weight component (M/sub r/ about 17,000). The low-molecular-weight protein was obtained in a homogeneous state by chromatography on phenyl-Sepharose. It was established that its properties are similar to those of calmodulin. The presence of calmodulin in preparations of phosphorylase kinase was judged by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in much the same way as bovine brain calmodulin. The results obtained suggest that the δ-subunit is a protein inhibitor of the enzyme

  13. ACQUISITION AND LOSS OF NEURONAL CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE DURING NEURONAL DIFFERENTIATION

    Science.gov (United States)

    Neurons display characteristic schedules by which they acquire and lose the neuron-specific Ca2+/calmodulin-dependent protein Kinase-Gr (CaM Kinase-Gr) during differentiation. uch schedules are exemplified by patterns of expression of this kinase in the developing cerebellum and ...

  14. Real-time Analysis of the Interaction between Calmodulin and Melittin by SPR Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The dynamic interaction process of calmodulin with an immobilized peptide melittin was investigated in real time by surface plasmon resonance spectroscopy, and dissociation constant of the complex was calculated to be 3.37′10-6 mol/L.

  15. Structural analysis of calmodulin binding to ion channels demonstrates the role of its plasticity in regulation.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Waterbeemd, M. van de; Bokhovchuk, F.M.; Bate, N.; Bindels, R.J.M.; Hoenderop, J.G.J.; Vuister, G.W.

    2013-01-01

    The Ca2+-binding protein calmodulin (CaM) is a well-known regulator of ion-channel activity. Consequently, the Protein Data Bank contains many structures of CaM in complex with different fragments of ion channels that together display a variety of binding modes. In addition to the canonical interact

  16. The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties.

    NARCIS (Netherlands)

    Kovalevskaya, N.V.; Bokhovchuk, F.M.; Vuister, G.W.

    2012-01-01

    The epithelial Ca(2+) channels TRPV5/6 (transient receptor potential vanilloid 5/6) are thoroughly regulated in order to fine-tune the amount of Ca(2+) reabsorption. Calmodulin has been shown to be involved into calcium-dependent inactivation of TRPV5/6 channels by binding directly to the distal C-t

  17. Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

    DEFF Research Database (Denmark)

    Li, Hongbing; Panina, Svetlana; Kaur, Amandeep;

    2012-01-01

    Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca2+/CaM complexes, which interact with and activate target proteins. ...

  18. A dynamic model of interactions of Ca2+, calmodulin, and catalytic subunits of Ca2+/calmodulin-dependent protein kinase II.

    Directory of Open Access Journals (Sweden)

    Shirley Pepke

    2010-02-01

    Full Text Available During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII, are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic

  19. Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2002-05-31

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. PMID:11919178

  20. Modulation of chloroplast movement in the green alga Mougeotia by the Ca2+ ionophore A23187 and by calmodulin antagonists.

    OpenAIRE

    Serlin, B S; Roux, S J

    1984-01-01

    The Ca2+ ionophore A23187 can induce chloroplast rotation within a single nonirradiated Mougeotia cell. The induced turning was dependent on the position of ionophore application and Ca2+ in the external medium. The role of calmodulin in mediating light-induced chloroplast rotation in the alga Mougeotia was investigated by using the paired calmodulin-antagonist drugs W5-W7 and W12-W13. In each pair, the antagonist with the greater affinity for calmodulin had the greater inhibitor effect on...

  1. Calmodulin mediates sulfur mustard toxicity in human keratinocytes

    International Nuclear Information System (INIS)

    Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48 h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural

  2. Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure

    Directory of Open Access Journals (Sweden)

    Hayashi Nobuhiro

    2008-02-01

    Full Text Available Abstract Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure

  3. Calmodulin as a major calcium buffer shaping vesicular release and short-term synaptic plasticity: facilitation through buffer dislocation

    Directory of Open Access Journals (Sweden)

    Yulia Timofeeva

    2015-07-01

    Full Text Available Action potential-dependent release of synaptic vesicles and short-term synaptic plasticity are dynamically regulated by the endogenous Ca2+ buffers that shape [Ca2+] profiles within a presynaptic bouton. Calmodulin is one of the most abundant presynaptic proteins and it binds Ca2+ faster than any other characterized endogenous neuronal Ca2+ buffer. Direct effects of calmodulin on fast presynaptic Ca2+ dynamics and vesicular release however have not been studied in detail. Using experimentally constrained three-dimensional diffusion modeling of Ca2+ influx–exocytosis coupling at small excitatory synapses we show that, at physiologically relevant concentrations, Ca2+ buffering by calmodulin plays a dominant role in inhibiting vesicular release and in modulating short-term synaptic plasticity. We also propose a novel and potentially powerful mechanism for short-term facilitation based on Ca2+-dependent dynamic dislocation of calmodulin molecules from the plasma membrane within the active zone.

  4. Purification and characterization of a Ca2+ -dependent/calmodulin-stimulated protein kinase from moss chloronema cells

    Indian Academy of Sciences (India)

    Jacinta S D’souza; Man Mohan Johri

    2003-03-01

    We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the moss Funaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 M free Ca2+, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Under in vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.

  5. Calmodulin and the target size of the (Ca2+ + Mg2+)-ATPase of human red-cell ghosts.

    Science.gov (United States)

    Cavieres, J D

    1984-04-11

    An average target size of 251 kDa has been obtained for the (Ca2+ + Mg2+)-ATPase of calmodulin-depleted erythrocyte ghosts by radiation inactivation with 16 MeV electrons. This is close to twice the size of the purified calcium-pump polypeptide. When calmodulin was included during the ATPase assay, a component of about 1 MDa appeared in addition to the activated dimer. PMID:6142728

  6. Calmodulin as a major calcium buffer shaping vesicular release and short-term synaptic plasticity: facilitation through buffer dislocation

    OpenAIRE

    Yulia Timofeeva; Kirill Volynski

    2015-01-01

    Action potential-dependent release of synaptic vesicles and short-term synaptic plasticity are dynamically regulated by the endogenous Ca(2+) buffers that shape [Ca(2+)] profiles within a presynaptic bouton. Calmodulin is one of the most abundant presynaptic proteins and it binds Ca(2+) faster than any other characterized endogenous neuronal Ca(2+) buffer. Direct effects of calmodulin on fast presynaptic Ca(2+) dynamics and vesicular release however have not been studied in detail. Using expe...

  7. Biosensor-Based Approach Identifies Four Distinct Calmodulin-Binding Domains in the G Protein-Coupled Estrogen Receptor 1

    OpenAIRE

    Tran, Quang-Kim; VerMeer, Mark

    2014-01-01

    The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific liga...

  8. Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues

    Directory of Open Access Journals (Sweden)

    Sixt SU

    2010-10-01

    Full Text Available Abstract The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved protein 1. Ubiquitylated proteins were degraded by the 26S protea-some in an ATP-depended manner. The degradation of ubiquitylated proteins were controlled by isopeptidase cleavage. A well characterised system of ubiquitylation and deubiquitylation is the calmodulin system in vitro 2. Detection of ubiquityl-calmodulin conjugtates in vivo have not been shown so far. In this article we discuss the detection of ubiquitin calmodulin conjugates in vivo by incubation with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues. Proteins with a molecular weight of ubiquityl-calmodulin conjugates could be detected in all organs tested. Incubation with ubiquitylprotein-isopeptidase showed clearly a decrease of ubiquitin calmodulin conjugates in vivo with an origination of unbounded ubiquitin. These results suggest that only few ubiquitin calmodulin conjugates exist in rabbit tissues.

  9. Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: More evidence for oxidative stress in vitiligo

    International Nuclear Information System (INIS)

    Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10-3 M H2O2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10-3M H2O2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H2O2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H2O2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo

  10. Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites.

    Science.gov (United States)

    Simon, Bertrand; Huart, Anne-Sophie; Temmerman, Koen; Vahokoski, Juha; Mertens, Haydyn D T; Komadina, Dana; Hoffmann, Jan-Erik; Yumerefendi, Hayretin; Svergun, Dmitri I; Kursula, Petri; Schultz, Carsten; McCarthy, Andrew A; Hart, Darren J; Wilmanns, Matthias

    2016-06-01

    The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation. PMID:27133022

  11. Distribution of calmodulin in corn seedlings - Immunocytochemical localization in coleoptiles and root apices

    Science.gov (United States)

    Dauwalder, M.; Roux, S. J.

    1986-01-01

    Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in etiolated corn (Zea mays, var. Bear Hybrid) seedlings. Uniform staining was seen in the background cytoplasm of most cell types. Cell walls and vacuoles were not stained. In coleoptile mesophyll cells the nucleoplasm of most nuclei was stained as was the stroma of most amyloplasts. The lumen border of mature tracheary elements in coleoptiles also stained. In the rootcap the most intensely stained regions were the cytoplasms of columella cells and of the outermost cells enmeshed in the layer of secreted slime. Nuclei in the rootcap cells did not stain distinctly, but those in all cell types of the root meristem did. Also in the root meristem, the cytoplasm of metaxylem elements stained brightly. These results are compared and contrasted with previous data on the localization of calmodulin in pea root apices and epicotyls and discussed in relation to current hypotheses on mechanisms of gravitropism.

  12. Cloning and Structural Analysis of Calmodulin Gene from the Mangrove Plant Sonneratia Paracaseolaris

    Institute of Scientific and Technical Information of China (English)

    Xiong Lingyuan; Lin Tao; Zhou Hantao; Xu Jinsen; Ge Yunsheng; Chen Muchuan; Chen Liang

    2002-01-01

    Calmodulin is a calcium binding protein that modulates the activity of diverse groups of protein including some protein kinase, adenylate cyclases and ATPase. Here we use the total DNA of Sonneratiaparacaseolaris as the template ofthe polymerase chain reaction (PCR). The PCR primers have been designed and synthesized according to the 5-and 3-terminal oligonucleotide sequences of Calmodulin gene of plants in Genbank and ligated with cloning vector pBsk(+).The recombinant clones have been obtained from the selected medium. The results of DNA sequences analysis show that the nucleotide sequences of ORF share more than 85% homologies as compared with those ofcalmodulin genes of several other plants. Similar to rice and apple, the ORF is interrupted by an intron behind the 75th nucleotide.

  13. Calmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis.

    Science.gov (United States)

    O'Day, Danton H; Myre, Michael A

    2004-08-01

    The calcium hypothesis of Alzheimer's disease (AD) invokes the disruption of calcium signaling as the underlying cause of neuronal dysfunction and ultimately apoptosis. As a primary calcium signal transducer, calmodulin (CaM) responds to cytosolic calcium fluxes by binding to and regulating the activity of target CaM-binding proteins (CaMBPs). Ca(2+)-dependent CaMBPs primarily contain domains (CaMBDs) that can be classified into motifs based upon variations on the basic amphiphilic alpha-helix domain involving conserved hydrophobic residues at positions 1-10, 1-14 or 1-16. In contrast, an IQ or IQ-like domain often mediates Ca(2+)-independent CaM-binding. Based on these attributes, a search for CaMBDs reveals that many of the proteins intimately linked to AD may be calmodulin-binding proteins, opening new avenues for research on this devastating disease. PMID:15249195

  14. Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels

    OpenAIRE

    Meissner, Gerhard; Pasek, Daniel A.; Yamaguchi, Naohiro; Ramachandran, Srinivas; Dokholyan, Nikolay V.; Tripathy, Ashutosh

    2009-01-01

    The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [35S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca2+ concentrations from

  15. AKAP150-mediated TRPV1 sensitization is disrupted by calcium/calmodulin

    OpenAIRE

    Shapiro Mark S; Belugin Sergei; Bal Manjot; Chaudhury Sraboni; Jeske Nathaniel A

    2011-01-01

    Abstract Background The transient receptor potential vanilloid type1 (TRPV1) is expressed in nociceptive sensory neurons and is sensitive to phosphorylation. A-Kinase Anchoring Protein 79/150 (AKAP150) mediates phosphorylation of TRPV1 by Protein Kinases A and C, modulating channel activity. However, few studies have focused on the regulatory mechanisms that control AKAP150 association with TRPV1. In the present study, we identify a role for calcium/calmodulin in controlling AKAP150 associati...

  16. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

    OpenAIRE

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2013-01-01

    Myosin XXI is the only myosin isoform expressed in the Leishmania parasite. The myosin-XXI homozygous knockout is lethal, and a reduction in expression levels leads to loss of endocytosis and affects other intracellular trafficking processes. In this paper we show that myosin XXI can adopt a monomeric or dimeric state. The states are determined by calmodulin binding to an IQ motif that, when bound, prevents dimerization of a coiled-coil motif. In the monomeric state the motor binds phospholip...

  17. Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain.

    Science.gov (United States)

    Harper, J F; Huang, J F; Lloyd, S J

    1994-06-14

    CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor. PMID:8003490

  18. Molecular cloning and characterization of a calmodulin-dependent phosphodiesterase enriched in olfactory sensory neurons.

    OpenAIRE

    C. Yan; Zhao, A Z; Bentley, J K; Loughney, K; Ferguson, K; Beavo, J. A.

    1995-01-01

    The sensing of an odorant by an animal must be a rapid but transient process, requiring an instant response and also a speedy termination of the signal. Previous biochemical and electrophysiological studies suggest that one or more phosphodiesterases (PDEs) may play an essential role in the rapid termination of the odorant-induced cAMP signal. Here we report the molecular cloning, expression, and characterization of a cDNA from rat olfactory epithelium that encodes a member of the calmodulin-...

  19. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    OpenAIRE

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII e...

  20. Calmodulin kinase II is required for angiotensin II-mediated vascular smooth muscle hypertrophy

    OpenAIRE

    Li, Hui; Li, Weiwei; Arun K Gupta; Mohler, Peter J.; Anderson, Mark E.; Grumbach, Isabella M.

    2009-01-01

    Despite our understanding that medial smooth muscle hypertrophy is a central feature of vascular remodeling, the molecular pathways underlying this pathology are still not well understood. Work over the past decade has illustrated a potential role for the multifunctional calmodulin-dependent kinase CaMKII in smooth muscle cell contraction, growth, and migration. Here we demonstrate that CaMKII is enriched in vascular smooth muscle (VSM) and that CaMKII inhibition blocks ANG II-dependent VSM c...

  1. Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function

    OpenAIRE

    Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L.; Sheffield, Val C.; Golan, Hava; Parvari, Ruti

    2015-01-01

    Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion o...

  2. Comprehensive Behavioral Analysis of Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice

    OpenAIRE

    Takao, Keizo; Tanda, Koichi; Nakamura, Kenji; Kasahara, Jiro; Nakao, Kazuki; Katsuki, Motoya; Nakanishi, Kazuo; Yamasaki, Nobuyuki; Toyama, Keiko; Adachi, Minami; UMEDA, MASAHIRO; Araki, Tsutomu; Fukunaga, Kohji; Kondo, Hisatake; Sakagami, Hiroyuki

    2010-01-01

    Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain...

  3. Detachment of glycolytic enzymes from cytoskeleton of melanoma cells induced by calmodulin antagonists.

    Science.gov (United States)

    Glass-Marmor, L; Beitner, R

    1997-06-11

    Glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. We have previously found that different calmodulin antagonists decrease the levels of allosteric activators of glycolysis, and reduce ATP content and cell viability in B16 melanoma cells. Here we report of a novel, additional, mechanism of action of calmodulin antagonists in melanoma cells. We show that these drugs cause a detachment of the glycolytic enzymes, phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), from cytoskeleton of B16 melanoma cells. This effect was dose- and time-dependent, and preceded the decrease in cell viability. The detachment of glycolytic enzymes from cytoskeleton would reduce the provision of local ATP, in the vicinity of the cytoskeleton-membrane and would affect cytoskeleton structure. Since the cytoskeleton is being recognized as an important modulator of cell function, proliferation, differentiation and neoplasia, detachment of the glycolytic enzymes from cytoskeleton induced by calmodulin antagonists, as well as their reported inhibitory action on cell proliferation, make these drugs most promising agents in treatment of cancer. PMID:9218707

  4. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    Science.gov (United States)

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  5. Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize

    Science.gov (United States)

    Stinemetz, C. L.; Hasenstein, K. H.; Young, L. M.; Evans, M. L.

    1992-01-01

    We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 micromole) that did not affect root growth. Calmodulin antagonists (> or = 1 micromole) inhibited downward transport of label from 45Ca2+ across the caps of gravistimulated roots relative to the downward transport of 45Ca2+ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip (> or = 1 micromole) also decreased the relative downward movement of label from 3H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of 45Ca2+ in the root tip nor the upward movement of label from 3H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists > or = 1 micromole become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.

  6. Calmodulin-dependent protein kinases mediate calcium-induced slow motility of mammalian outer hair cells.

    Science.gov (United States)

    Puschner, B; Schacht, J

    1997-08-01

    Cochlear outer hair cells in vitro respond to elevation of intracellular calcium with slow shape changes over seconds to minutes ('slow motility'). This process is blocked by general calmodulin antagonists suggesting the participation of calcium/calmodulin-dependent enzymatic reactions. The present study proposes a mechanism for these reactions. Length changes of outer hair cells isolated from the guinea pig cochlea were induced by exposure to the calcium ionophore ionomycin. ATP levels remained unaffected by this treatment ruling out depletion of ATP (by activation of calcium-dependent ATPases) as a cause of the observed shape changes. Involvement of protein kinases was suggested by the inhibition of shape changes by K252a, a broad-spectrum inhibitor of protein kinase activity. Furthermore, the inhibitors ML-7 and ML-9 blocked the shape changes at concentrations compatible with inhibition of myosin light chain kinase (MLCK). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), also attenuated the length changes. Inhibitors with selectivity for cyclic nucleotide-dependent protein kinases (H-89, staurosporine) were tested to assess potential additional contributions by such enzymes. The dose dependence of their action supported the notion that the most likely mechanism of slow motility involves phosphorylation reactions catalyzed by MLCK or CaMKII or both. PMID:9282907

  7. Subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

    International Nuclear Information System (INIS)

    Incubation of subcellular fractions isolated from rat cerebral cortex with [gamma-32P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes

  8. Purification, crystallization and preliminary crystallographic studies of a calmodulin-OLFp hybrid molecule

    International Nuclear Information System (INIS)

    The hybrid moelcule of calmodulin and calmodulin-binding domain of olfactory nucleotide-gated ion-channel peptide (CaM-OLFp) was crystallized and preliminary analyzed using X-ray diffaction. A hybrid molecule consisting of calmodulin (CaM) and the CaM-binding domain of olfactory nucleotide-gated ion-channel peptide (CaM-OLFp) was purified and crystallized by the hanging-drop vapour-diffusion method at 298 K. The crystals diffracted to a maximum resolution of 1.85 Å at cryogenic temperature (100 K) using X-rays from a rotating anode (Cu, wavelength 1.54 Å). The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 64.76, b = 36.23, c = 70.96 Å, α = γ = 90, β = 109.4°. Analysis of the packing density shows that the asymmetric unit contains one CaM-OLFp hybrid molecule with a solvent content of 36.42%

  9. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Science.gov (United States)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  10. Biosensor-based approach identifies four distinct calmodulin-binding domains in the G protein-coupled estrogen receptor 1.

    Directory of Open Access Journals (Sweden)

    Quang-Kim Tran

    Full Text Available The G protein-coupled estrogen receptor 1 (GPER has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca(2+-elevating agent. To confirm direct interaction and locate the calmodulin-binding domain(s, we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER's submembrane domains (SMDs. Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83-93, 150-175, 242-259, 330-351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca(2+-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent K d values being 0.44 ± 0.03, 1.40 ± 0.16, 8.01 ± 0.29, and 136.62 ± 6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca(2+ indicators identified separate Ca(2+ sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca(2+ response, representing two distinct species (SMD1 sp1 and SMD1 sp2 with drastically different Ca(2+ sensitivities. The Ca(2+ sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca(2+ values being 0.13 ± 0.02, 0.75 ± 0.05, 2.38 ± 0.13, 3.71 ± 0.13, and 5.15 ± 0.25 µM, respectively. These data indicate that calmodulin may regulate GPER

  11. A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.

    Science.gov (United States)

    Mukherjee, Somnath; Ura, Marcin; Hoey, Robert J; Kossiakoff, Anthony A

    2015-08-14

    Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD~2 pM), which can be reversed by EDTA allowing controlled "capture and release" of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. KD values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks. PMID:26159704

  12. Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode*

    OpenAIRE

    Ishida, Hiroaki; Rainaldi, Mario; Vogel, Hans J.

    2009-01-01

    The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca2+-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca2+-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of b...

  13. Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulin-dependent protein kinase homolog

    Science.gov (United States)

    Lu, Y. T.; Feldman, L. J.

    1997-01-01

    Roots of many species grow downward (orthogravitropism) only when illuminated. Previous work suggests that this is a calcium-regulated response and that both calmodulin and calcium/calmodulin-dependent kinases participate in transducing gravity and light stimuli. A genomic sequence has been obtained for a calcium/calmodulin-dependent kinase homolog (MCK1) expressed in root caps, the site of perception for both light and gravity. This homolog consists of 7265 base pairs and contains 11 exons and 10 introns. Since MCK1 is expressed constitutively in both light and dark, it is unlikely that the light directly affects MCK1 expression, though the activity of the protein may be affected by light. In cultivars showing light-regulated gravitropism, we hypothesize that MCK1, or a homolog, functions in establishing the auxin asymmetry necessary for orthogravitropism.

  14. Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Poovaiah, B. W.

    2002-01-01

    Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.

  15. Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

    Science.gov (United States)

    Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

    1999-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

  16. Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

    International Nuclear Information System (INIS)

    Inositol 1,4,5-triphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, the authors describe the partial purification of the 3-kinase from the cytosolic fraction of bovine brain and studies of its catalytic properties. They found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, they propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, and intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [γ-32P]ATP by thin-layer chromatography. Using this new assay method, they evaluated kinetic parameters (K/sub m/ for ATP = 40 μM, K/sub m/ for Ins-1,4,5-P3 = 0.7 μM, K/sub i/ for ADP = 12 μM) and divalent cation specificity (Mg2+ > > Mn2+ > Ca2+) for the 3-kinase

  17. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses

    OpenAIRE

    Heo, Won Do; Lee, Sang Hyoung; Kim, Min Chul; Kim, Jong Cheol; Chung, Woo Sik; Chun, Hyun Jin; Lee, Kyoung Joo; Park, Chan Young; Park, Hyeong Cheol; Choi, Ji Young; Cho, Moo Je

    1999-01-01

    The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, wher...

  18. The Ca2+/Calmodulin-Dependent Protein Kinase Kinase, CaMKK2, Inhibits Preadipocyte Differentiation

    OpenAIRE

    Lin, Fumin; Ribar, Thomas J.; Means, Anthony R.

    2011-01-01

    When fed a standard chow diet, CaMKK2 null mice have increased adiposity and larger adipocytes than do wild-type mice, whereas energy balance is unchanged. Here, we show that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is expressed in preadipocytes, where it functions as an AMP-activated protein kinase (AMPK)α kinase. Acute inhibition or deletion of CaMKK2 in preadipocytes enhances their differentiation into mature adipocytes, which can be reversed by 5-aminoimidazole-4-carboxa...

  19. Role of calcium and calmodulin in reaction of gastric fundus contraction

    Directory of Open Access Journals (Sweden)

    Marta Gajdus

    2011-09-01

    Full Text Available Background:The subject of this study is determination of the influence of calmodulin and calcium on gastric fundus smooth muscle contraction. During experiments, the author tested the influence of a serotonin receptor agonist, serotonin (5-HT, causing smooth muscle contraction.Material/Methods:Testing was conducted on tissues isolated from rat’s stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg. The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification.Results:According to conducted tests, we can deduce that 8 Br cGMP stops the reaction of gastric fundus smooth muscle contraction induced by serotonin. The use of 8Br-cGMP in the range of concentrations between 10 and 300 µM leads to reduction of maximum effect from 100�0to 46�20Similar changes were obtained after the use of guanylate cyclase activator (CG – YC-1. Curves for the contractile activity of serotonin along with an increase of concentration YC-1 are shifted to the right, and the maximum effect of reaction decreases. Increasing concentrations of flunarizine, a calmodulin antagonist, in a concentration-dependent way blocks binding between calcium and calmodulin, and at the same time leads to the shift of concentration-effect curves for serotonin to the right and a decrease of maximum reaction.Increasing concentrations of ODQ, a guanylate cyclase inhibitor lead to statistically significant shift of the curves to the left, decrease of EC50 value and simultaneous increase of maximum reaction to serotonin.Conclusions:According to conducted testing, serotonin causes gastric fundus smooth muscle contraction dependent on

  20. Regulation of Calcium/Calmodulin-dependent Kinase IV by O-GlcNAc Modification*

    OpenAIRE

    Dias, Wagner B.; Cheung, Win D.; Wang, Zihao; Hart, Gerald W.

    2009-01-01

    Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV ...

  1. Clicked bis-PEG-peptide conjugates for studying calmodulin-Kv7.2 channel binding

    OpenAIRE

    Bonache de Marcos, María Ángeles; Alaimo, Alessandro; Malo, Covadonga; Millet, Oscar; Villarroel, Alvaro; González-Muñiz, Rosario

    2014-01-01

    The recombinant Kv7.2 calmodulin (CaM) binding site (Q2AB CaMBD) shows a high tendency to aggregate, thus complicating biochemical and structural studies. To facilitate these studies we have conceived bis-PEG-peptide CaMBD-mimetics linking helices A and B in single, easy to handle molecules. Short PEG chains were selected as spacers between the two peptide molecules, and a Cu(i)-catalyzed cycloaddition (CuAAC) protocol was used to assemble the final bis-PEG-peptide conjugate, by the convenien...

  2. Characterization of Novel Calmodulin Binding Domains within IQ Motifs of IQGAP1

    OpenAIRE

    Jang, Deok-Jin; Ban, Byungkwan; Lee, Jin-A

    2011-01-01

    IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca2+-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM bind...

  3. Calmodulin and S100A1 Protein Interact with N Terminus of TRPM3 Channel

    Czech Academy of Sciences Publication Activity Database

    Holakovská, Blanka; Gryčová, Lenka; Jirků, Michaela; Šulc, Miroslav; Bumba, Ladislav; Teisinger, Jan

    2012-01-01

    Roč. 287, č. 20 (2012), s. 16645-16655. ISSN 0021-9258 R&D Projects: GA ČR(CZ) GAP301/10/1159; GA ČR(CZ) GPP205/10/P308; GA ČR(CZ) GAP207/11/0717; GA ČR(CZ) GD305/03/H148 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50200510 Keywords : TRPM3 * calmodulin * S100A1 * surface plasmon resonance * fluorescence anisotropy Subject RIV: BO - Biophysics Impact factor: 4.651, year: 2012

  4. Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

    OpenAIRE

    Bosser, R; Faura, M; Serratosa, J; Renau-Piqueras, J; Pruschy, M; Bachs, O

    1995-01-01

    It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nucl...

  5. Retinoic acid inhibits calmodulin binding to human erythrocyte membranes and reduces membrane Ca2(+)-adenosine triphosphatase activity.

    OpenAIRE

    Davis, F B; Smith, T. J.; Deziel, M R; Davis, P J; Blas, S D

    1990-01-01

    Ca2(+)-ATPase activity in human red cell membranes is dependent on the presence of calmodulin. All trans-retinoic acid inhibited human red cell membrane Ca2(+)-ATPase activity in vitro in a concentration-dependent manner (10(-8) to 10(-4) M). In contrast, retinol, retinal, 13-cis-retinoic acid and the benzene ring analogue of retinoic acid did not alter enzyme activity. Purified calmodulin (up to 500 ng/ml, 3 X 10(-8) M) added to red cell membranes, in the presence of inhibitory concentration...

  6. Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

    Science.gov (United States)

    Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Giorgetti, Alejandro; Dominici, Paola; Astegno, Alessandra

    2016-08-01

    In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. PMID:27124620

  7. Isolation of Hybridomas for Golgi-associated Proteins and a Plant Calmodulin

    Science.gov (United States)

    Kuzmanoff, K. M.; Ray, P. M.

    1985-01-01

    The demonstration of a role for calcium in the mechanism of the gravitropic response indicates a role for calmodulin. Localization studies indicate that plant cell walls have a high content of calmodulin which suggests a regulatory role for CaM in both gravitropic curvature and auxin-induced growth. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the ctivity of Golgi-localized B-1,4-glucan synthase (GS), an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. In order to determine whether auxin stimulates GS activity either by modulation of existing enzyme or induces de novo formation of Golgi glucan synthase, a study was undertaken to isolate and quantitate glucan synthase. This enzyme appears to be an integral protein of the Golgi membrane and has resisted isolation with retention of activity. The production of monoclonal antibody for glucan synthase was undertaken due to the inability to isolate GS by standard detergent/liposome techniques.

  8. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    OpenAIRE

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic,...

  9. SPLICE VARIANT SPECIFIC UPREGULATIONOF CA+2/CALMODULIN DEPENDENT PROTEIN KINASE 1G BY PYRETHROID INSECTICIDES IN VIVO.

    Science.gov (United States)

    Pyrethroid insecticides induce neurotoxicity in mammals by interfering with ion channel function in excitable neuronal membranes. Previous work demonstrated dose-dependent increases in expression of Ca+2/calmodulin dependent protein kinase (Camk1g) mRNA following acute deltameth...

  10. Ca2+-calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Kiens, Bente; Richter, Erik

    2006-01-01

    Ca2+ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed...

  11. Intracellular transduction in the regulation of pheromone biosynthesis of the silkworm, Bombyx mori: suggested involvement of calmodulin and phosphoprotein phosphatase.

    Science.gov (United States)

    Matsumoto, S; Ozawa, R; Nagamine, T; Kim, G H; Uchiumi, K; Shono, T; Mitsui, T

    1995-03-01

    We have tested the effects of chemicals on bombykol production in vitro in the silkworm, Bombyx mori, to probe the biochemical steps as well as underlying mechanisms regulated by PBAN. These results suggest the involvement of calmodulin and phosphoprotein phosphatase in the intracellular signal transduction of PBAN action. PMID:7766202

  12. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    Science.gov (United States)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  13. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  14. Detection of calmodulin binding protein at 170 KDA in BALB, AKR, DON and chicken granulosa cells

    International Nuclear Information System (INIS)

    Calmodulin (CAM) has been shown to bind to the epidermal growth factor (EGF) receptor (170 kDa) and is phosphorylated in a EGF dependent manner in the A431 human epidermoid carcinoma cells. In the present study, they report 125I-CAM binding to a 170 kDa protein detected in cell membrane vesicles of Balb/3T3, AKR, DON and chicken granulosa cells. Purified plasma membranes from these cells were resolved via electrophoresis (without heat denaturation) and electroblotted onto nictrocellulose paper. Upon hybridizing against 125I-CAM, a distinct autoradiographic band occurred at 170 kDa for all the cells lines under study. The binding of CAM is specific and can be displaced with the addition of excess unlabeled CAM. The result suggest that 125I-CAM may bind to the 170 kDa EGF receptor in BALB, AKR, DON and chicken granulosa cells

  15. Immunohistochemical locali- zation of Ca2+/calmodulin- dependent kinase in tobacco

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The existence of Ca2+/calmodulin-dependent kinase (CaM kinase, CaMK) in tobacco is verified immuno- logically and its distribution in different tissues of tobacco is studied. It has been demonstrated that CaMK is mainly distributed in early developing anthers, developing ovules and embryos, lateral root primordium, apical meristem and leaf primordium of buds and mesophyll cells and developing vascular bundles of leaves. There is enormous CaM kinase distributed in leaf epidermis fair cells and guard cells of stomas too. Little kinase is found in mature stem or root cells. The distribution properties of CaM kinase in tobacco are consistent with those of CaM, suggesting that there exists the Ca2+ signal transduction pathway mediated by CaM kinase in tobacco and it plays an important role in the plant growth and development.

  16. Assembly and Calcium Binding Properties of Quantum Dot-Calmodulin Calcium Sensor.

    Science.gov (United States)

    Eun, Su-yong; Nguyen-ta, Kim; Yoo, Hoon; Silva, Gabriel A; Kim, Soon-jong

    2016-02-01

    We have developed the first nanoengineered quantum dot molecular complex designed to measure changes of calcium ion (Ca2+) concentration at high spatial and temporal resolutions in real time. The sensor is ratiometric and composed of three components: a quantum dot (QD) emitting at 620 nm as a fluorescence donor, an organic dye (Alexa Fluor 647) as a fluorescence acceptor, and a calmodulin-M13 (CaM-M13) protein part as a calcium sensing component. In this work, we have determined the maximal number of CaM-M13 required for saturating a single QD particle to be approximately 16. The dissociation constant, Kd of the QD-based calcium ion sensor was also estimated to be around 30 microM. PMID:27433729

  17. Interaction of a plant pseudo-response regulator with a calmodulin-like protein

    Energy Technology Data Exchange (ETDEWEB)

    Perochon, Alexandre; Dieterle, Stefan; Pouzet, Cecile; Aldon, Didier; Galaud, Jean-Philippe [UMR 5546 CNRS/Universite Toulouse 3, Pole de Biotechnologie vegetale, BP 42617 Auzeville, 31326 Castanet-Tolosan cedex (France); Ranty, Benoit, E-mail: ranty@scsv.ups-tlse.fr [UMR 5546 CNRS/Universite Toulouse 3, Pole de Biotechnologie vegetale, BP 42617 Auzeville, 31326 Castanet-Tolosan cedex (France)

    2010-08-06

    Research highlights: {yields} The pseudo-response regulator PRR2 specifically binds CML9, a calmodulin-like protein {yields} The interaction is confirmed in plant cell nuclei {yields} The interaction requires an intact PRR2 protein. -- Abstract: Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

  18. Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

    International Nuclear Information System (INIS)

    Highlights: → Both Ca++-Calmodulin (CaM) and Ca++-free CaM bind to the C-terminal region of Nav1.1. → Ca++ and CaM have both opposite and convergent effects on INav1.1. → Ca++-CaM modulates INav1.1 amplitude. → CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. → Ca++ alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca++ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca++ could bind the Nav1.1 C-terminal region with micromolar affinity.

  19. Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

    Energy Technology Data Exchange (ETDEWEB)

    Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Clare, Jeffrey J. [Eaton Pharma Consulting, Eaton Socon, Cambridgeshire PE19 8EF (United Kingdom); Debanne, Dominique [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Alcaraz, Gisele, E-mail: gisele.alcaraz@univmed.fr [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France)

    2011-07-29

    Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.

  20. Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response through calmodulin-binding transcription factor AtSR1/CAMTA3.

    Science.gov (United States)

    Qiu, Yongjian; Xi, Jing; Du, Liqun; Suttle, Jeffrey C; Poovaiah, B W

    2012-05-01

    Calcium/calmodulin (Ca(2+)/CaM) has long been considered a crucial component in wound signaling pathway. However, very few Ca(2+)/CaM-binding proteins have been identified which regulate plant responses to herbivore attack/wounding stress. We have reported earlier that a family of Ca(2+)/CaM-binding transcription factors designated as AtSRs (also known as AtCAMTAs) can respond differentially to wounding stress. Further studies revealed that AtSR1/CAMTA3 is a negative regulator of plant defense, and Ca(2+)/CaM-binding to AtSR1 is indispensable for the suppression of salicylic acid (SA) accumulation and disease resistance. Here we report that Ca(2+)/CaM-binding is also critical for AtSR1-mediated herbivore-induced wound response. Interestingly, atsr1 mutant plants are more susceptible to herbivore attack than wild-type plants. Complementation of atsr1 mutant plants by overexpressing wild-type AtSR1 protein can effectively restore plant resistance to herbivore attack. However, when mutants of AtSR1 with impaired CaM-binding ability were overexpressed in atsr1 mutant plants, plant resistance to herbivore attack was not restored, suggesting a key role for Ca(2+)/CaM-binding in wound signaling. Furthermore, it was observed that elevated SA levels in atsr1 mutant plants have a negative impact on both basal and induced biosynthesis of jasmonates (JA). These results revealed that Ca(2+)/CaM-mediated signaling regulates plant response to herbivore attack/wounding by modulating the SA-JA crosstalk through AtSR1. PMID:22371088

  1. 2,5-hexanedione (HD) treatment alters calmodulin, Ca2+/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

    International Nuclear Information System (INIS)

    Calcium-dependent mechanisms, particularly those mediated by Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50 mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC and SN were significantly increased, which indicated elevation of Ca2+ concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs

  2. Neutron and x-ray scattering studies of the interactions between Ca{sup 2+}-binding proteins and their regulatory targets: Comparisons of troponin C and calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J.; Olah, G.A.

    1993-11-01

    The regulatory proteins calmodulin and troponin C share a strikingly unusual overall structure. Their crystal structures show each protein consists of two structurally homologous globular domains connected by an extended, solvent exposed alpha-helix of = 8 turns. Calmodulin regulates a variety of enzymes that show remarkable functional and structural diversity. This diversity extends to the amino acid sequences of the calmodulin-binding domains in the target enzymes. In contrast with calodulin, troponin C appears to have a single very specialized function. It is an integral part of the troponin complex, and Ca{sup 2+} binding to troponin c results in the release of the inhibitory function of troponin I, which eventually leads to actin-binding to myosin and the triggering of muscle contraction. Small-angle scattering has been particularly useful for studying the dumbbell shaped proteins because the technique is very sensitive to changes in the relative dispositions of the two globular domains. Small-angle scattering, using x-rays or neutrons, gives information on the overall shapes of proteins in solution. Small-angle scattering studies of calmodulin and its complexes with calmodulin-binding domains from various target enzymes have played an important role in helping us understand the functional role of its unusual solvent exposed helix. Likewise, small-angle scattering has been used to study troponin C with various peptides, to shed light on the similarities and differences between calmodulin and troponin C.

  3. Distinct Properties of \\(Ca^{2+}\\)-Calmodulin Binding to N- and C-Terminal Regulatory Regions of the TRPV1 Channel

    OpenAIRE

    Gaudet, Rachelle; Lau, Sze-Yi; Procko, Erik

    2012-01-01

    Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TR...

  4. Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

    DEFF Research Database (Denmark)

    Shi, Juan; Geshi, Naomi; Takahashi, Shinichi;

    2013-01-01

    The molecular mechanism underlying Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cel...... essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells....

  5. Dendritic spine changes in the development of alcohol addiction regulated by α-calcium/calmodulin-dependent protein kinase II

    OpenAIRE

    Zofia Mijakowska

    2014-01-01

    Introduction Alcohol has many adverse effects on the brain. Among them are dendritic spine morphology alterations, which are believed to be the basis of alcohol addiction. Autophosphorylation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII) has been shown to regulate spine morphology in vitro. Here we show that αCaMKII can also regulate addiction related behaviour and dendritic spine morphology changes caused by alcohol consumption in vivo. Method 12 αCaMKII-autophosphorylatio...

  6. Simulation of Ca-Calmodulin-Dependent Protein Kinase II on Rabbit Ventricular Myocyte Ion Currents and Action Potentials

    OpenAIRE

    Grandi, Eleonora; Puglisi, Jose L.; Wagner, Stefan; Maier, Lars S.; Severi, Stefano; Bers, Donald M.

    2007-01-01

    Ca-calmodulin-dependent protein kinase II (CaMKII) was recently shown to alter Na+ channel gating and recapitulate a human Na+ channel genetic mutation that causes an unusual combined arrhythmogenic phenotype in patients: simultaneous long QT syndrome and Brugada syndrome. CaMKII is upregulated in heart failure where arrhythmias are common, and CaMKII inhibition can reduce arrhythmias. Thus, CaMKII-dependent channel modulation may contribute to acquired arrhythmic disease. We developed a Mark...

  7. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

    OpenAIRE

    Glaser, P; Elmaoglou-Lazaridou, A; Krin, E.; Ladant, D.; Bârzu, O; Danchin, A

    1989-01-01

    In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture sup...

  8. Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

    OpenAIRE

    Tadross, Michael R.; Johny, Manu Ben; Yue, David T.

    2010-01-01

    Ca2+/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca2+ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation i...

  9. Ocular dominance plasticity is stably maintained in the absence of α calcium calmodulin kinase II (αCaMKII) autophosphorylation

    OpenAIRE

    Sharif A Taha; Stryker, Michael P.

    2005-01-01

    The molecule α calcium calmodulin kinase II (αCaMKII) is known to play a fundamental role in the induction of many forms of synaptic plasticity. A major theory of αCaMKII function proposes that autophosphorylation of the molecule mediates not only the induction but also the maintenance of synaptic plasticity. To test this hypothesis, we assessed ocular dominance plasticity in genetically engineered mice that carry a mutation preventing autophosphorylation of αCaMKII. These mutant mice are def...

  10. Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    International Nuclear Information System (INIS)

    We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca2+ leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca2+/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca2+ transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca2+ transients results in the appearance of trains of Ca2+ spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca2+ events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.

  11. Isolation and characterization of a novel calmodulin-binding protein from potato

    Science.gov (United States)

    Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

    2002-01-01

    Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

  12. A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

    Science.gov (United States)

    Safadi, F.; Reddy, V. S.; Reddy, A. S.

    2000-01-01

    Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

  13. The structure of the complex of calmodulin with KAR-2: a novel mode of binding explains the unique pharmacology of the drug.

    Science.gov (United States)

    Horváth, István; Harmat, Veronika; Perczel, András; Pálfi, Villo; Nyitray, László; Nagy, Attila; Hlavanda, Emma; Náray-Szabó, Gábor; Ovádi, Judit

    2005-03-01

    3'-(beta-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca(2+)-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation ((1)H-(15)N heteronuclear single quantum coherence) spectra of (15)N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets. PMID:15596444

  14. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    Science.gov (United States)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  15. Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane.

    Science.gov (United States)

    Alli, Abdel A; Bao, Hui-Fang; Liu, Bing-Chen; Yu, Ling; Aldrugh, Summer; Montgomery, Darrice S; Ma, He-Ping; Eaton, Douglas C

    2015-09-01

    Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane. PMID:26136560

  16. Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans

    KAUST Repository

    Chen, Zhangfan

    2012-08-01

    The polychaete . Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of . H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of . H. elegans. A full-length . CaM cDNA was successfully cloned from . H. elegans (. He-CaM) and it contained an open reading frame of 450. bp, encoding 149 amino acid residues. It was highly expressed in 12. h post-metamorphic juveniles, and remained high in adults. . In situ hybridization conducted in competent larvae and juveniles revealed that . He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that . He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of . H. elegans. © 2012 Elsevier Inc..

  17. Cloning and analysis of calmodulin gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

    Science.gov (United States)

    Wang, Mengqiang; Mao, Yunxiang; Zhuang, Yunyun; Kong, Fanna; Sui, Zhenghong

    2009-09-01

    In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

  18. The ever changing moods of calmodulin: how structural plasticity entails transductional adaptability.

    Science.gov (United States)

    Villarroel, Alvaro; Taglialatela, Maurizio; Bernardo-Seisdedos, Ganeko; Alaimo, Alessandro; Agirre, Jon; Alberdi, Araitz; Gomis-Perez, Carolina; Soldovieri, Maria Virginia; Ambrosino, Paolo; Malo, Covadonga; Areso, Pilar

    2014-07-29

    The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca(2+). Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca(2+) loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca(2+) conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca(2+) binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure-function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca(2+)-dependent and Ca(2+)-independent signals. PMID:24857860

  19. Cllmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines.

    Science.gov (United States)

    Haußer, I; Herth, W; Reiss, H D

    1984-09-01

    Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming. PMID:24253945

  20. Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release.

    Science.gov (United States)

    Taylor, James E; Chow, John Y H; Jeffries, Cy M; Kwan, Ann H; Duff, Anthony P; Hamilton, William A; Trewhella, Jill

    2012-08-01

    Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ~70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca(2+) or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca(2+)-signaling during viral processing. PMID:22947870

  1. The Ca(2+ influence on calmodulin unfolding pathway: a steered molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    Full Text Available The force-induced unfolding of calmodulin (CaM was investigated at atomistic details with steered molecular dynamics. The two isolated CaM domains as well as the full-length CaM were simulated in N-C-terminal pulling scheme, and the isolated N-lobe of CaM was studied specially in two other pulling schemes to test the effect of pulling direction and compare with relevant experiments. Both Ca(2+-loaded CaM and Ca(2+-free CaM were considered in order to define the Ca(2+ influence to the CaM unfolding. The results reveal that the Ca(2+ significantly affects the stability and unfolding behaviors of both the isolated CaM domains and the full-length CaM. In Ca(2+-loaded CaM, N-terminal domain unfolds in priori to the C-terminal domain. But in Ca(2+-free CaM, the unfolding order changes, and C-terminal domain unfolds first. The force-extension curves of CaM unfolding indicate that the major unfolding barrier comes from conquering the interaction of two EF-hand motifs in both N- and C- terminal domains. Our results provide the atomistic-level insights in the force-induced CaM unfolding and explain the observation in recent AFM experiments.

  2. Subtle pH differences trigger single residue motions for moderating conformations of calmodulin

    CERN Document Server

    Atilgan, Ali Rana; Atilgan, Canan

    2011-01-01

    This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca2+ signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations to analyze conformational preferences of calcium-loaded CaM, initially in extended form. PRS is comprised of sequential application of directed forces on residues followed by recording the resulting coordinates. We show that manipulation of a single residue, E31 located in one of the EF hand motifs, reproduces structural changes to compact forms, and the flexible linker acts as a transducer of binding information to distant parts of the protein. Independently, using four different pKa calculation strategies, we find E31...

  3. Calmodulin effects on steroids-regulated plasma membrane calcium pump activity.

    Science.gov (United States)

    Zylinska, Ludmila; Kowalska, Iwona; Ferenc, Bozena

    2009-03-01

    It is now generally accepted that non-genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca(2+) is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca(2+) increase is ATP-consuming plasma membrane calcium pump (PMCA). The enzyme is coded by four genes, but isoforms diversity was detected in excitable and non-excitable cells. It is the only ion pump stimulated directly by calmodulin (CaM). We examined the role of PMCA isoforms composition and CaM effect in regulation of Ca(2+) uptake by estradiol, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and their sulfates in a concentration range from 10(-9) to 10(-6) M, using the membranes from rat cortical synaptosomes, differentiated PC12 cells, and human erythrocytes. In excitable membranes with full set of PMCAs steroids apparently increased Ca(2+) uptake, although to a variable extent. In most of the cases, CaM decreased transport by 30-40% below controls. Erythrocyte PMCA was regulated by the steroids somewhat differently than excitable cells. CaM strongly increased the potency for Ca(2+) extrusion in membranes incubated with 17-beta-estradiol and PREG. Our results indicated that steroids may sufficiently control cytoplasmic calcium concentration within physiological and therapeutic range. The response depended on the cell type, PMCA isoforms expression profile, CaM presence, and the steroids structure. PMID:19226536

  4. Calmodulin activation by calcium transients in the postsynaptic density of dendritic spines.

    Directory of Open Access Journals (Sweden)

    Daniel X Keller

    Full Text Available The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.

  5. Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

    Institute of Scientific and Technical Information of China (English)

    WANG Mengqiang; MAO Yunxiang; ZHUANG Yunyun; KONG Fanna; SUI Zhenghong

    2009-01-01

    In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensiss,cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exous and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.

  6. Responses of plant calmodulin to endocytosis induced by rare earth elements.

    Science.gov (United States)

    Wang, Lihong; Cheng, Mengzhu; Chu, Yunxia; Li, Xiaodong; Chen, David D Y; Huang, Xiaohua; Zhou, Qing

    2016-07-01

    The wide application of rare earth elements (REEs) have led to their diffusion and accumulation in the environment. The activation of endocytosis is the primary response of plant cells to REEs. Calmodulin (CaM), as an important substance in calcium (Ca) signaling systems, regulating almost all of the physiological activities in plants, such as cellular metabolism, cell growth and division. However, the response of CaM to endocytosis activated by REEs remains unknown. By using immunofluorescence labeling and a confocal laser scanning microscope, we found that trivalent lanthanum [La(III)], an REE ion, affected the expression of CaM in endocytosis. Using circular dichroism, X-ray photoelectron spectroscopy and computer simulations, we demonstrated that a low concentration of La(III) could interact with extracellular CaM by electrostatic attraction and was then bound to two Ca-binding sites of CaM, making the molecular structure more compact and orderly, whereas a high concentration of La(III) could be coordinated with cytoplasmic CaM or bound to other Ca-binding sites, making the molecular structure more loose and disorderly. Our results provide a reference for revealing the action mechanisms of REEs in plant cells. PMID:27081794

  7. Calcium/calmodulin-dependent protein kinase IV: A multifunctional enzyme and potential therapeutic target.

    Science.gov (United States)

    Naz, Huma; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-05-01

    The calcium/calmodulin-dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family, and is primarily involved in transcriptional regulation in lymphocytes, neurons and male germ cells. CAMKIV operates the signaling cascade and regulates activity of several transcription activators by phosphorylation, which in turn plays pivotal roles in immune response, inflammation and memory consolidation. In this review, we tried to focus on different aspects of CAMKIV to understand the significance of this protein in the biological system. This enzyme is associated with varieties of disorders such as cerebral hypoxia, azoospermia, endometrial and ovarian cancer, systemic lupus, etc., and hence it is considered as a potential therapeutic target. Structure of CAMKIV is comprised of five distinct domains in which kinase domain is responsible for enzyme activity. CAMKIV is involved in varieties of cellular functions such as regulation of gene expression, T-cell maturation, regulation of survival phase of dendritic cells, bone growth and metabolism, memory consolidation, sperm motility, regulation of microtubule dynamics, cell-cycle progression and apoptosis. In this review, we performed an extensive analysis on structure, function and regulation of CAMKIV and associated diseases. PMID:26773169

  8. Photounbinding of calmodulin from a family of CaM binding peptides.

    Directory of Open Access Journals (Sweden)

    Klaus G Neumüller

    Full Text Available BACKGROUND: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. METHODOLOGY/PRINCIPAL FINDINGS: We present studies of the photounbinding of labeled calmodulin (CaM from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. CONCLUSIONS/SIGNIFICANCE: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.

  9. Molecular characteristics and expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii.

    Science.gov (United States)

    Zeng, L-G; Wang, J-H; Li, Y-J; Sheng, J-Q; Gu, Q; Hong, Y-J

    2012-01-01

    Calmodulin (CaM) is a multifunctional intracellular calcium ion receptor protein that participates in a range of cellular processes, including calcium metabolism in mussels. To investigate the role of CaM in freshwater mollusk shell calcium metabolism, the full-length CaM cDNA was isolated from the freshwater pearl mussel, Hyriopsis schlegelii (referred to as hsCaM) using SMART RACE technology. The full-length hsCaM was 855 bp in size, containing a 70-bp 5'-untranslated sequence, a 447-bp open reading frame, a 309-bp 3'-untranslated sequence, and a 26-nucleotide long poly(A) tail. The hsCaM mRNA expression in different mussel tissues was examined using real-time PCR. The hsCaM mRNA was found to be ubiquitously expressed, but far more abundant in the gill, foot, and mantle than in the posterior adductor muscle. Real-time PCR was also used to determine hsCaM mRNA expression levels in mantle tissues of H. schlegelii at different ages. No significant differences between one-, two-, and three-year-old mussels were detected, but expression increased in four-year-old mussels and then decreased in five-year-old mussels. CaM appears to be involved in calcium regulation of the mantle in four-year-old mussels, which may secrete more mother of pearl during pearl culture. PMID:22290464

  10. PTPRZ1 regulates calmodulin phosphorylation and tumor progression in small-cell lung carcinoma

    International Nuclear Information System (INIS)

    Small-cell lung carcinoma (SCLC) is a neuroendocrine tumor subtype and comprises approximately 15% of lung cancers. Because SCLC is still a disease with a poor prognosis and limited treatment options, there is an urgent need to develop targeted molecular agents for this disease. We screened 20 cell lines from a variety of pathological phenotypes established from different organs by RT-PCR. Paraffin-embedded tissue from 252 primary tumors was examined for PTPRZ1 expression using immunohistochemistry. shRNA mediated PTPRZ1 down-regulation was used to study impact on tyrosine phosphorylation and in vivo tumor progression in SCLC cell lines. Here we show that PTPRZ1, a member of the protein tyrosine- phosphatase receptor (PTPR) family, is highly expressed in SCLC cell lines and specifically exists in human neuroendocrine tumor (NET) tissues. We also demonstrate that binding of the ligand of PTPRZ1, pleiotrophin (PTN), activates the PTN/PTPRZ1 signaling pathway to induce tyrosine phosphorylation of calmodulin (CaM) in SCLC cells, suggesting that PTPRZ1 is a regulator of tyrosine phosphorylation in SCLC cells. Furthermore, we found that PTPRZ1 actually has an important oncogenic role in tumor progression in the murine xenograft model. PTPRZ1 was highly expressed in human NET tissues and PTPRZ1 is an oncogenic tyrosine phosphatase in SCLCs. These results imply that a new signaling pathway involving PTPRZ1 could be a feasible target for treatment of NETs

  11. High-level expression of human calmodulin in E. coli and its effects on cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Xiao Jun Li; Jian Guo Wu; Jun Ling Si; Da Wen Guo; Jian Ping Xu

    2000-01-01

    Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1,2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.

  12. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

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    Grant S. Nichols

    2015-01-01

    Full Text Available Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX, the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults.

  13. Distinguishing Unfolding and Functional Conformational Transitions of Calmodulin Using Ultraviolet Resonance Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jones, Eric M.; Balakrishnan, G.; Squier, Thomas C.; Spiro, Thomas

    2014-06-14

    Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium-binding protein exhibits a broad range of structural transitions, including domain opening and folding-unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well known, it has been challenging to examine the dynamics of transition between these structures in real-time, due to a lack of suitable spectroscopic probes of CaM structure. In this paper, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C-terminal domain in response to Ca2+ binding. This spectral difference is entirely due to differences in teritary contacts at the inter-domain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation-sensitive tyrosine residues, opening a path to time-resolved studies of CaM dynamics using Raman spectroscopy.

  14. Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation.

    Science.gov (United States)

    Reichow, Steve L; Gonen, Tamir

    2008-09-10

    Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation. PMID:18786401

  15. Progress in the participation of Ca2+-calmodulin in heat shock signal transduction

    Institute of Scientific and Technical Information of China (English)

    Rengang Zhou; Bing Li; Hongtao Liu; Daye Sun

    2009-01-01

    A novel heat shock (HS) signal transduction pathway in plants for the participation of Ca2+-calmodulin (CAM) in HS signal trans-duction was identified. HS induces a rapid increase in intracellular free calcium ion levels ([Ca2+]i), and the involvement of phospholipase C-inositol 1,4,5-trisphosphate is one of the factors leading to elevation in [Ca2+]i induced by HS. HS also increases the expression of the CaM gene and the accumulation of the CaM protein. The CaM isoform, AtCaM3, in Arabidopsis is a key member in the HS signal trans-duction pathway. AtCaM3 regulates the activity of CaM-binding protein kinase (AtCBK3) or protein phosphatase (AtPP7), promoting the activation of the HS transcription factor, AtHSFA1a, by phosphorylation/dephosphorylation and the expression of heat shock pro-tein genes, then improving heat tolerance in plants.

  16. Growth, Gas Exchange, Abscisic Acid, and Calmodulin Response to Salt Stress in Three Poplars

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In the present study, we investigated the effects of increasing salinity on growth, gas exchange, abscisic acid(ABA), calmodulin (CAM), and the relevance to salt tolerance in seedlings of Populus euphratica Oliv. and cuttings of P. "pupularis 35-44" (P. popularis) and P. x euramericana cv. 1-214 (P. cv. Italica). The relative growth rates of shoot height (RGRH) for P. cv. Italica and P. popularis were severely reduced by increasing salt stress,whereas the growth reduction was relatively less in P. euphratica. Similarly, P. euphratica maintained higher net photosynthetic rates (Pn) and unit transpiration rates (TRN) than P. cv. Italica and P. popularis under conditions of higher salinity. Salinity caused a significant increase in leaf ABA and CaM in the three genotypes after the onset of stress, but NaCl-induced ABA and CaM accumulation was more pronounced in P. euphratica,suggesting that P. euphratica plants are more sensitive in sensing soil salinity than the other two poplars.Furthermore, P. euphratica maintained relatively higher ABA and CaM concentrations under conditions of high salinity. The higher capacity to synthesize stress signals, namely ABA and CaM, in P. euphratica and the contribution of this to the salt resistance of P. euphratica are discussed.

  17. Comparing allosteric transitions in the domains of calmodulin through coarse-grained simulations

    CERN Document Server

    Nandigrami, Prithviraj

    2015-01-01

    Calmodulin (CaM) is a ubiquitous calcium binding protein consisting of two structurally similar domains with distinct stabilities, binding affinities, and flexibilities. We present coarse grained simulations that suggest the mechanism for the domain's allosteric transitions between the open and closed conformations depend on subtle differences in the folded state topology of the two domains. Throughout a wide temperature range, the simulated transition mechanism of the N-terminal domain (nCaM) follows a two-state transition mechanism while domain opening in the C-terminal domain (cCaM) involves unfolding and refolding of the tertiary structure. The appearance of the unfolded intermediate occurs at a higher temperature in nCaM than it does in cCaM. That is, we find that cCaM unfolds more readily along the transition route than nCaM. Furthermore, unfolding and refolding of the domain significantly slows the domain opening and closing rates of cCaM, a distinct scenario which can potentially influence the mechani...

  18. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Bækgaard, Lone;

    2010-01-01

    of calcium-bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The......Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example...... crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 A, beta = 113.2 degrees. A complete data set was collected to 3.0 A resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin....

  19. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    KAUST Repository

    Chen, Zhang-Fan

    2012-02-13

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus (= Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca 2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. © 2012 Chen et al.

  20. Ca(2+)-Calmodulin regulation of testicular androgen production in Mozambique tilapia (Oreochromis mossambicus).

    Science.gov (United States)

    Martins, Rute S T; Fuentes, Juan; Almeida, Olinda; Power, Deborah M; Canario, Adelino V M

    2009-06-01

    The Ca(2+)-Calmodulin (CaM) signaling pathway has previously been shown to be involved in the regulation of teleost fish ovarian steroidogenesis. However, a putative role of CaM in testicular steroidogenesis and potential targets has not been examined. To examine whether basal steroidogenesis is modulated by Ca(2+) and CaM levels in the testis of Mozambique tilapia (Oreochromis mossambicus) we have incubated testicular fragments in vitro under different conditions and analyzed steroid output. Calcium-free medium with or without EGTA did not affect testicular basal 11-ketotestosterone (11-KT) and testosterone (T) secretion. However, addition of 80microM the CaM inhibitor W7 significantly reduced basal 11-KT, T and androstenedione secretion. Interestingly, the decreased androgen production by 80microM of W7 was accompanied by increased 11-desoxicortisol output and by the activation of cortisol synthesis in the testis, the latter undetected in untreated tissues. However, production of 17,20alpha-dihydroxy-4-pregnen-3-one was unaltered by W7. This suggests that C17,20 desmolase, 21-hydroxylase and possibly 11beta-hydroxysteroid dehydrogenase are targets for CaM. In addition, androgen production was also found to be regulated by the level of cAMP since incubations with forskolin (FK) significantly increased 11-KT and T output. A cross-talk between the cAMP and Ca(2+)-CaM signaling pathways was detected since W7 administration also decreased FK stimulated androgen production. Altogether, these data show that both basal and cAMP stimulated androgen levels were modulated by intracellular Ca(2+)-dependent CaM and that possibly Ca(2+)-CaM determines the shift in steroidogenesis from C21 steroids to androgens. PMID:19341736

  1. α-Calcium calmodulin kinase II modulates the temporal structure of hippocampal bursting patterns.

    Directory of Open Access Journals (Sweden)

    Jeiwon Cho

    Full Text Available The alpha calcium calmodulin kinase II (α-CaMKII is known to play a key role in CA1/CA3 synaptic plasticity, hippocampal place cell stability and spatial learning. Additionally, there is evidence from hippocampal electrophysiological slice studies that this kinase has a role in regulating ion channels that control neuronal excitability. Here, we report in vivo single unit studies, with α-CaMKII mutant mice, in which threonine 305 was replaced with an aspartate (α-CaMKII(T305D mutants, that indicate that this kinase modulates spike patterns in hippocampal pyramidal neurons. Previous studies showed that α-CaMKII(T305D mutants have abnormalities in both hippocampal LTP and hippocampal-dependent learning. We found that besides decreased place cell stability, which could be caused by their LTP impairments, the hippocampal CA1 spike patterns of α-CaMKII(T305D mutants were profoundly abnormal. Although overall firing rate, and overall burst frequency were not significantly altered in these mutants, inter-burst intervals, mean number of intra-burst spikes, ratio of intra-burst spikes to total spikes, and mean intra-burst intervals were significantly altered. In particular, the intra burst intervals of place cells in α-CaMKII(T305D mutants showed higher variability than controls. These results provide in vivo evidence that besides its well-known function in synaptic plasticity, α-CaMKII, and in particular its inhibitory phosphorylation at threonine 305, also have a role in shaping the temporal structure of hippocampal burst patterns. These results suggest that some of the molecular processes involved in acquiring information may also shape the patterns used to encode this information.

  2. Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease.

    Science.gov (United States)

    Vassilakopoulou, Vyronia; Calver, Brian L; Thanassoulas, Angelos; Beck, Konrad; Hu, Handan; Buntwal, Luke; Smith, Adrian; Theodoridou, Maria; Kashir, Junaid; Blayney, Lynda; Livaniou, Evangelia; Nounesis, George; Lai, F Anthony; Nomikos, Michail

    2015-11-01

    Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca(2+) channel complex that mediates Ca(2+) efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation-contraction coupling and defective CaM-RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca(2+)-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca(2+)-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca(2+)-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [(3)H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca(2+)-binding as well as defective interaction with RyR2. PMID:26164367

  3. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    Science.gov (United States)

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  4. Cooperative phenomena in binding and activation of Bordetella pertussis adenylate cyclase by calmodulin.

    Science.gov (United States)

    Bouhss, A; Krin, E; Munier, H; Gilles, A M; Danchin, A; Glaser, P; Bârzu, O

    1993-01-25

    The catalytic domain of Bordetella pertussis adenylate cyclase located within the first 400 amino acids of the protein can be cleaved by trypsin in two subdomains (T25 and T18) corresponding to ATP-(T25) and calmodulin (CaM)-(T18) binding sites. Reassociation of subdomains by CaM is a cooperative process, which is a unique case among CaM-activated enzymes. To understand better the molecular basis of this phenomenon, we used several approaches such as partial deletions of the adenylate cyclase gene, isolation of peptides of various size, and site-directed mutagenesis experiments. We found that a stretch of 72 amino acid residues overlapping the carboxyl terminus of T25 and the amino terminus of T18 accounts for 90% of the binding energy of adenylate cyclase-CaM complex. The hydrophobic "side" of the helical region situated around Trp242 plays a major role in the interaction of adenylate cyclase with CaM, whereas basic residues that alternate with acidic residues in bacterial enzyme play a much less important role. The amino-terminal half of the catalytic domain of adenylate cyclase contributes only 10% to the binding energy of CaM, whereas the last 130 amino acid residues are not at all involved in binding. However, these segments of adenylate cyclase might affect protein/protein interaction and catalysis by propagating conformational changes to the CaM-binding sequence which is located in the middle of the catalytic domain of bacterial enzyme. PMID:8420945

  5. Human platelet calmodulin-binding proteins: Ca/sup 2 +/-dependent proteolysis upon platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1986-05-01

    Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and /sup 125/I-CaM. Ten distinct proteins with molecular weights of 245, 225K, 175K, 150K, 90K, 82K(2), 60K and 41K(2) bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner; the binding was blocked by both trifluoperazine and nonradiolabeled CaM. The 225K and 90K proteins were labeled by antisera against myosin light chain kinase (MLCK); the 60K and one of the 82K proteins were identified as the CaM-dependent phosphatase and caldesmon. The remaining proteins have not yet been identified. Most of the CaM-binding proteins were degraded upon addition of Ca/sup 2 +/ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin or N-ethyl-maleimide which suggests that it was due to a Ca/sup 2 +/-dependent protease. Activation of intact platelets by thrombin, ADP, collagen and the Ca/sup 2 +/-ionophores A23187 and ionomycin under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca/sup 2 +/) also resulted in limited proteolysis of CaM-binding proteins including those labeled with anti-MLCK and the phosphatase. Many Ca/sup 2 +//CaM-regulated enzymes have been shown to be irreversibly activated in vitro by limited proteolysis. Their data indicates that limited proteolysis also occurs in vivo; under certain conditions proteolysis may be an important physiological mechanism for irreversibly activating Ca/sup 2 +//CaM-regulated enzymes.

  6. Oxidized calmodulin kinase II regulates conduction following myocardial infarction: a computational analysis.

    Directory of Open Access Journals (Sweden)

    Matthew D Christensen

    2009-12-01

    Full Text Available Calmodulin kinase II (CaMKII mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ. These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.

  7. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Hudmon, Andy; Schulman, Howard

    2002-06-15

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a 'molecular switch-like' behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure-function of this multifunctional protein kinase. PMID:11931644

  8. Regulation of K-Ras4B Membrane Binding by Calmodulin.

    Science.gov (United States)

    Sperlich, Benjamin; Kapoor, Shobhna; Waldmann, Herbert; Winter, Roland; Weise, Katrin

    2016-07-12

    K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell. PMID:27410739

  9. Isolation and Characterization of Calmodulin Gene of Alexandrium catenella (Dinoflagellate) and Its Performance in Cell Growth and Heat Stress

    Institute of Scientific and Technical Information of China (English)

    WEN Ruobing; SUI Zhenghong; BAO Zhenmin; ZHOU Wei; WANG Chunyan

    2014-01-01

    Harmful algal blooms (HABs) can occur and then disappear quickly, corresponding to consistent growing and declining of heavy biomasses. The molecular mechanism of blooming remains unclear. In this study, calmodulin gene (cam) of HAB causing species Alexandrium catenella was isolated and characterized. The expression of calmodulin gene was profiled at different growth rates and in heat stress. The full cDNA of cam was 597 nucleotides (nt) in length, including a 25 nt 5′untranslated region (UTR), an 122 nt 3′ UTR, and a 450 nt open reading frame (ORF) encoding 149 amino acids. The deduced calmodulin (CaM) was highly conserved in comparison with those of other organisms. As was determined with real-time RT PCR, the abundance of cam transcript varied in a pattern similar to cell growth rate during the whole growing period. The abundance of cam transcript increased by more than 8 folds from lag growth phase to exponential growth phase, and then obviously decreased from exponential growth phase to stationary/decline growth phase. In addition, the relative abundance of cam transcript significantly declined with time during heat shock. Taking CaM function described in other organisms into account, we believe that Ca2+-involved signal transduction, methyla-tion of DNA and toxin precursors underlined the cell growth of this species. The response of cam gene to heat stress in dinoflagellate suggested restrictions in Ca2+signal transduction and methylation. These findings are helpful to understand the relationships among growth, cell signal transduction, bloom formation and interaction with environmental stimuli in dinoflagellates.

  10. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

    International Nuclear Information System (INIS)

    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K+ (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM−1 s−1 and 2.55 ± 1.50 s−1, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies

  11. Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

    Science.gov (United States)

    Huang, J. F.; Teyton, L.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

  12. Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation.

    Directory of Open Access Journals (Sweden)

    Peter Rellos

    Full Text Available UNLABELLED: Long-term potentiation (LTP, a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM-dependent kinase II (CaMKII. CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIdelta/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIdelta/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix alphaD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the Web plugin are available in Text S1.

  13. Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries

    DEFF Research Database (Denmark)

    Waldsee, Roya; Ahnstedt, Hilda; Eftekhari, Sajedeh;

    2010-01-01

    Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ET(B) receptors in smooth muscle cells via increased transcription. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CAMK......(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ET(A) and ET(B) receptors and CAMKII were determined by real-time PCR, and their protein levels were evaluated by immunohistochemistry and Western blot. The mRNA levels of CAMKII and the ET(B) receptor increased during organ...

  14. Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner

    Czech Academy of Sciences Publication Activity Database

    Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

    2012-01-01

    Roč. 7, č. 1 (2012), e30529. E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GA204/07/1592; GA ČR(CZ) GD204/09/H084; GA ČR GAP305/11/2232; GA MŠk LC545; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : NM1 * nuclear import * NLS * calmodulin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

  15. Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport

    DEFF Research Database (Denmark)

    Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion;

    2006-01-01

    Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d...... response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux....

  16. Calmodulin Kinase II Interacts with the Dopamine Transporter C Terminus to Regulate Amphetamine-Induced Reverse Transport

    DEFF Research Database (Denmark)

    Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion;

    2006-01-01

    Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d...... response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux....

  17. Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.

    OpenAIRE

    Tippetts, M T; Robertson, D L

    1988-01-01

    The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like th...

  18. Long-Term Memory Deficits in Pavlovian Fear Conditioning in Ca2+/Calmodulin Kinase Kinase α-Deficient Mice▿

    OpenAIRE

    Blaeser, Frank; Sanders, Matthew J.; Truong, Nga; Ko, Shanelle; Wu, Long Jun; Wozniak, David F.; Fanselow, Michael S.; Zhuo, Min; Chatila, Talal A.

    2006-01-01

    Signaling by the Ca2+/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase α (CaMKKα) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKα mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context...

  19. Regulating effect of calcium ion,calmodulin and calmodulin dependent protein kinase in opioid addiction%钙离子及其结合蛋白、蛋白激酶对阿片成瘾的调节作用

    Institute of Scientific and Technical Information of China (English)

    张静

    2015-01-01

    The calcium ion is an important intracellular second messenger. The calcium ion,calmodulin and calmodulin dependent protein kinase play an important role in drug dependence,with the interaction with the mesolimbic dopamine system. Further study on calcium ion will lead to expand the understanding of the neural mechanisms underlying drug addiction,and provide an effective way for the development of low tolerance,low dependence of pain medicine as well as for the treatment of opioid addiction and relapse.%钙离子是细胞内重要的第二信使。与受体偶联的细胞内钙离子、钙调蛋白和两者依赖的钙蛋白激酶Ⅱ能够通过与中脑边缘多巴胺系统的相互作用来影响药物成瘾,在药物依赖和耐受等过程中具有重要作用。对三者的研究将指引人们对药物成瘾过程神经机制的理解,并为开发低耐受性、低依赖性镇痛药物和药物依赖的防治以及解决戒毒后的“复吸”等问题奠定基础。

  20. Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

    Directory of Open Access Journals (Sweden)

    J.B.R. Rodriguez

    2013-03-01

    Full Text Available Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endoplasmic reticulum Ca2+-ATPase isoform (SERCA is expressed in rat vas deferens (RVD and its modulation by calmodulin (CaM-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM and a low sensitivity to vanadate (IC50 = 41 µM. These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

  1. Inhibitors of the Ca2+/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

    International Nuclear Information System (INIS)

    Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca2+/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro

  2. Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

    Science.gov (United States)

    Lu, Y. T.; Hidaka, H.; Feldman, L. J.

    1996-01-01

    Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

  3. Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

    International Nuclear Information System (INIS)

    Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity

  4. Using a GFP-gene fusion technique to study the cell cycle-dependent distribution of calmodulin in living cells

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

    In this study, a green fluorescent protein (GFP)-calmodulin (CaM) fusion gene method was used to examine the distribution of calmodulin during various stages of cell cycle. First, it was found that the distribution of CaM in living cells changes with the cell cycle. CaM was found mainly in the cytoplasm during G1 phase. It began to move into the nucleus when the cell entered S phase. At G2 phase, CaM became more concentrated in the nucleus than in cytoplasm. Second, the accumulation of CaM in the nucleus during G2 phase appeared to be related to the onset of mitosis, since inhibiting the activation of CaM at this stage resulted in blocking the nuclear membrane breakdown and chromatin condensation. Finally, after the cell entered mitosis, a high concentration of CaM was found at the polar regions of the mitotic spindle. At this time, inhibiting the activity of CaM would cause a disruption of the spindle structure. The relationship between the stage-specific distribution of CaM and its function in regulat

  5. Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies.

    Science.gov (United States)

    Kuczera, Krzysztof; Kursula, Petri

    2012-01-01

    We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the

  6. Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase.

    Science.gov (United States)

    Gribovskaja, Irena; Brownlow, Kaleb C; Dennis, Sam J; Rosko, Andrew J; Marletta, Michael A; Stevens-Truss, Regina

    2005-05-24

    Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation. PMID:15896003

  7. Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

    Directory of Open Access Journals (Sweden)

    Sitvanit Haziza

    2015-08-01

    Full Text Available Calmodulin lysine methyl transferase (CaM KMT is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence

  8. Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

    Science.gov (United States)

    Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L; Sheffield, Val C; Golan, Hava; Parvari, Ruti

    2015-08-01

    Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the Ca

  9. Identification of the Calmodulin-Binding Domains of Fas Death Receptor.

    Directory of Open Access Journals (Sweden)

    Bliss J Chang

    Full Text Available The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD and that of the Fas-associated protein (FADD interact to form the core of the death-inducing signaling complex (DISC, a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD. However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR, biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1 and 251-288 (Fas-Pep2 constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling

  10. Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein.

    Science.gov (United States)

    O'Day, Danton H; Chatterjee-Chakraborty, Munmun; Wagler, Stephanie; Myre, Michael A

    2005-06-17

    Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein. PMID:15883042

  11. Upregulation of c-FLIP-short in response to TRAIL promotes survival of NSCLC cells, which could be suppressed by inhibition of Ca2+/calmodulin signaling

    Czech Academy of Sciences Publication Activity Database

    Kaminskyy, V.O.; Surova, O.V.; Piskunova, T.; Zborovskaya, I.B.; Tchevkina, E.M.; Anděra, Ladislav; Zhivotovsky, B.

    2013-01-01

    Roč. 4, březen 2013 (2013), e522. ISSN 2041-4889 Institutional support: RVO:68378050 Keywords : TRAIL * DR4 * c-FLIPS * calcium * calmodulin * lung adenocarcinoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.177, year: 2013

  12. Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system

    DEFF Research Database (Denmark)

    Panina, Svetlana; Stephan, Alexander; la Cour, Jonas M;

    2012-01-01

    Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model...

  13. Purification and partial characterization of a novel calcium-binding protein from Bacillus cereus T spores and inhibition of germination by calmodulin antagonists

    International Nuclear Information System (INIS)

    A novel calcium-binding protein has been purified from the dormant spores of Bacillus cereus T. B. cereus T spores were extensively washed, broken, and heated at 90 degree C for 2 min. Using calcium-dependent hydrophobic interaction chromatography plus DEAE-cellulose and hydroxylapatite columns, a single protein was obtained which possessed calcium-binding capacity and some characteristics of calmodulin. This heat-stable protein was retained by hydrophobic matrices or a calmodulin antagonist in a calcium-dependent manner. The crude spore extract displaced bovine brain calmodulin from its antibody in a radioimmunoassay and the immunoreactive specific activity of the partially purified fraction which eluted from phenyl-Sepharose was ca. 200-fold greater than the crude spore extract. Purity of this protein was verified by sodium dodecyl sulfate-polyarcylamide gel electrophoresis and reversed-phase HPLC. Calcium-binding ability was verified with a competitive calcium binding assay using Chelex-100 resin and 45Ca autoradiography. SDS-PAGE and amino acid composition indicated the molecular weight of the protein was 24-kDa. The effects of two calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) on L-alanine-induced germination of Bacillus cereus T spores were examined by measuring commitment to germination, loss of heat resistance, release of calcium, decrease in optical density at 660 nm and phase-contrast microscopy

  14. Isolation, characterization, and bioinformatic analysis of calmodulin-binding protein cmbB reveals a novel tandem IP22 repeat common to many Dictyostelium and Mimivirus proteins.

    Science.gov (United States)

    O'Day, Danton H; Suhre, Karsten; Myre, Michael A; Chatterjee-Chakraborty, Munmun; Chavez, Sara E

    2006-08-01

    A novel calmodulin-binding protein cmbB from Dictyostelium discoideum is encoded in a single gene. Northern analysis reveals two cmbB transcripts first detectable at 4 h during multicellular development. Western blotting detects an approximately 46.6 kDa protein. Sequence analysis and calmodulin-agarose binding studies identified a "classic" calcium-dependent calmodulin-binding domain (179IPKSLRSLFLGKGYNQPLEF198) but structural analyses suggest binding may not involve classic alpha-helical calmodulin-binding. The cmbB protein is comprised of tandem repeats of a newly identified IP22 motif ([I,L]Pxxhxxhxhxxxhxxxhxxxx; where h = any hydrophobic amino acid) that is highly conserved and a more precise representation of the FNIP repeat. At least eight Acanthamoeba polyphaga Mimivirus proteins and over 100 Dictyostelium proteins contain tandem arrays of the IP22 motif and its variants. cmbB also shares structural homology to YopM, from the plague bacterium Yersenia pestis. PMID:16777069

  15. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    Science.gov (United States)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  16. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    Science.gov (United States)

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

  17. Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin2.

    Science.gov (United States)

    Karlsson, Göran; Persson, Cecilia; Mayzel, Maxim; Hedenström, Mattias; Backman, Lars

    2016-04-01

    Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker. PMID:26800385

  18. Rat vas deferens SERCA2 is modulated by Ca{sup 2+}/calmodulin protein kinase II-mediated phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.B.R.; Muzi-Filho, H. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Valverde, R.H.F. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Quintas, L.E.M. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Noel, F. [Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Einicker-Lamas, M. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ (Brazil); Cunha, V.M.N. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2013-03-19

    Ca{sup 2+} pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca{sup 2+}-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca{sup 2+} (Ca{sub 0.5} = 780 nM) and a low sensitivity to vanadate (IC{sub 50} = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca{sup 2+} and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca{sup 2+} accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca{sup 2+} and CaM, possibly via CaMKII, in a process that results in stimulation of Ca{sup 2+} pumping activity.

  19. Kinetics of the inhibition of calcium/calmodulin-dependent protein kinase II by pea protein-derived peptides.

    Science.gov (United States)

    Li, Huan; Aluko, Rotimi E

    2005-11-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzes the phosphorylation of various cellular proteins and excessive activities have been implicated in the pathogenesis of various chronic diseases. We hypothesized that positively charged peptides can be produced through enzymatic hydrolysis of pea proteins; such peptides could then bind to negatively charged calmodulin (CaM) at a physiological pH level and inhibit CaMKII activity. Pea protein isolate was hydrolyzed with an alkaline protease (alcalase) and filtered through a 1000-mol wt cutoff membrane. The permeate, which contained low-molecular weight peptides, was used to isolate cationic peptides on an SP-Sepharose column by ion exchange chromatography. Separation of the permeate on the SP-Sepharose column yielded two fractions with net positive charges that were subsequently used for enzyme inhibition studies. Fraction I eluted earlier from the column and contained lower contents of lysine and arginine than Fraction II, which eluted later. Results show that both peptide fractions inhibited CaMKII activity mostly in a competitive manner, although kinetic data suggested that inhibition by Fraction II may be of the mixed type. Kinetic analysis (K(m) and K(i)) showed that affinity of peptides in Fraction II for CaM was more than that in Fraction I, which was directly correlated with the higher inhibitory properties of Fraction II against CaMKII. The results suggest that it may be possible to use pea protein-derived cationic peptides to modulate CaMKII activities. PMID:16111873

  20. Cross-talk between calcium-calmodulin and nitric oxide in abscisic acid signaling in leaves of maize plants

    Institute of Scientific and Technical Information of China (English)

    Jianrong Sang; Aying Zhang; Fan Lin; Mingpu Tan; Mingyi Jiang

    2008-01-01

    Using pharmacological and biochemical approaches,the signaling pathways between hydrogen peroxide (H2O2),calcium (Ca2+)-calmodulin (CAM),and nitric oxide (NO) in abscisic acid (ABA)-induced antioxidant defense were investigated in leaves of maize (Zea mays L.) plants.Treatments with ABA,H2O2,and CaCI2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase (NOS) in the cytosolic and microsomal fractions of maize leaves.However,such increases were blocked by the pretreatments with Ca2+ inhibitors and CaM antagonists.Meanwhile,pretreatments with two NOS inhibitors also suppressed the Ca2+-induced increase in the production of NO.On the other hand,treatments with ABA and the NO donor sodium nitroprusside (SNP) also led to increases in the concentration of cytosolic Ca2+ in protoplasts of mesophyll cells and in the expression of calmodulin 1 (CaMI) gene and the contents of CaM in leaves of maize plants,and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor.Moreover,SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 (SOD4),cytosolic ascorbate peroxidase (cAPX),and glutathione reductase 1 (GRI) and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca2+ inhibitors and CaM antagonists.Our results suggest that Ca2+-CaM functions both upstream and downstream of NO production,which is mainly from NOS,in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.

  1. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    Science.gov (United States)

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  2. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G; Pińna, L A

    1994-01-01

    The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability to...... substitute for wild-type beta-subunit as a suppressor of activity toward calmodulin. The only mutations that reduced the ability of the beta-subunit to suppress calmodulin phosphorylation activity, though being compatible with normal reconstitution of CK2 holoenzyme, were those affecting Asp55, Glu57 and the...... conversely ineffective. The latent "calmodulin kinase" activity of CK2 can also be specifically unmasked by a peptide (alpha[66-86]) reproducing a basic insert of the catalytic subunit. This effect is reversed by equimolar addition of a peptide (beta[55-71]) including the 55-64 acidic stretch of the beta...

  3. 钙调素-人胰岛素原融合体的酶切加工%ENZYMATIC DIGESTION OF CALMODULIN-PROINSULIN FUSION PROTEIN

    Institute of Scientific and Technical Information of China (English)

    王学祥; 井健

    2011-01-01

    A fusion protein composed of human proinsuiin mutant and calmodulin was prepared, then digested with PRESCI protease. Digested product includes calmodulin and human proinsulin mutant. Human proinsulin mutant was purified.%通过基因工程方法将人胰岛素原突变体偶联钙调索形成融合态重组蛋白质,在该重组蛋白质中设计具有PRESCI蛋白酶酶切位点.制备PRESCI蛋白酶并对钙调素-人胰岛素原突变体重组蛋白质进行酶切加工.研究表明,该重组融合蛋白质可被有效切割,通过蛋白质纯化手段能够有效地将切割后的钙调素与人胰岛素原突变体进行纯化.

  4. Phosphorylation and activation of nuclear Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca{sup 2+}/calmodulin-dependent protein kinase I (CaMKI)

    Energy Technology Data Exchange (ETDEWEB)

    Onouchi, Takashi [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Ishida, Atsuhiko [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521 (Japan); Kameshita, Isamu [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer CaMKP-N/PPM1E underwent proteolytic processing and translocated to cytosol. Black-Right-Pointing-Pointer The proteolysis was effectively inhibited by the proteasome inhibitors. Black-Right-Pointing-Pointer Ser-480 of zebrafish CaMKP-N was phosphorylated by cytosolic CaMKI. Black-Right-Pointing-Pointer Phosphorylation-mimic mutants of CaMKP-N showed enhanced activity. Black-Right-Pointing-Pointer These results suggest that CaMKP-N is regulated by CaMKI. -- Abstract: Nuclear Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca{sup 2+}/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.

  5. Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

    OpenAIRE

    Lakowicz, J.R.; Gryczynski, I.; Laczko, G; Wiczk, W; Johnson, M.L.

    1994-01-01

    We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distance...

  6. Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase

    Science.gov (United States)

    Vitart, V.; Christodoulou, J.; Huang, J. F.; Chazin, W. J.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

  7. Calcium/Calmodulin-dependent Protein Kinase II is a Ubiquitous Molecule in Human Long-term Memory Synaptic Plasticity: A Systematic Review

    OpenAIRE

    Negar Ataei; Ali Mohammad Sabzghabaee; Ahmad Movahedian

    2015-01-01

    Background: Long-term memory is based on synaptic plasticity, a series of biochemical mechanisms include changes in structure and proteins of brain′s neurons. In this article, we systematically reviewed the studies that indicate calcium/calmodulin kinase II (CaMKII) is a ubiquitous molecule among different enzymes involved in human long-term memory and the main downstream signaling pathway of long-term memory. Methods: All of the observational, case-control and review studies were conside...

  8. Calcium/calmodulin-dependent protein kinase II is a ubiquitous molecule in human long-term memory synaptic plasticity: A systematic review

    Directory of Open Access Journals (Sweden)

    Negar Ataei

    2015-01-01

    Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory.

  9. Targeting the Small- and Intermediate-Conductance Ca2+-Activated Potassium Channels: The Drug-Binding Pocket at the Channel/Calmodulin Interface

    Directory of Open Access Journals (Sweden)

    Meng Cui

    2014-10-01

    Full Text Available The small- and intermediate-conductance Ca2+-activated potassium (SK/IK channels play important roles in the regulation of excitable cells in both the central nervous and cardiovascular systems. Evidence from animal models has implicated SK/IK channels in neurological conditions such as ataxia and alcohol use disorders. Further, genome-wide association studies have suggested that cardiovascular abnormalities such as arrhythmias and hypertension are associated with single nucleotide polymorphisms that occur within the genes encoding the SK/IK channels. The Ca2+ sensitivity of the SK/IK channels stems from a constitutively bound Ca2+-binding protein: calmodulin. Small-molecule positive modulators of SK/IK channels have been developed over the past decade, and recent structural studies have revealed that the binding pocket of these positive modulators is located at the interface between the channel and calmodulin. SK/IK channel positive modulators can potentiate channel activity by enhancing the coupling between Ca2+ sensing via calmodulin and mechanical opening of the channel. Here, we review binding pocket studies that have provided structural insight into the mechanism of action for SK/IK channel positive modulators. These studies lay the foundation for structure-based drug discovery efforts that can identify novel SK/IK channel positive modulators. © 2014 S. Karger AG, Basel

  10. Graded Ca2+/calmodulin-dependent coupling of voltage-gated CaV1.2 channels

    Science.gov (United States)

    Dixon, Rose E; Moreno, Claudia M; Yuan, Can; Opitz-Araya, Ximena; Binder, Marc D; Navedo, Manuel F; Santana, Luis F

    2015-01-01

    In the heart, reliable activation of Ca2+ release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca2+ to calmodulin (CaM) and proceed through Ca2+/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. Our findings suggest a model for CaV1.2 channel gating and Ca2+-influx amplification that unifies diverse observations about Ca2+ signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently. DOI: http://dx.doi.org/10.7554/eLife.05608.001 PMID:25714924

  11. Development of a new Ca2+/calmodulin antagonist and its anti-proliferative activity against colorectal cancer cells

    International Nuclear Information System (INIS)

    We previously identified a cellular target of a cell cycle inhibitor HBC as Ca2+/calmodulin (Ca2+/CaM) through chemical genetics approach. Using the mechanism-based drug design, we developed a new Ca2+/CaM antagonists based on the structure of HBC. The compound, (4-{3,5-bis-[2-(4-hydroxy-3-methoxy-phenyl)-vinyl]-4,5-dihydro-pyrazol-1-yl }-phenyl)-(4-methyl-piperazin-1-yl)-methanone (referred as HBCP), binds to Ca2+/CaM in vitro and inhibits the proliferation of HCT15 colon cancer cells. HBCP induced sustained phosphorylation of ERK1/2 and subsequently activated p21WAF1 expression in HCT15 cells. Moreover, HBCP reversibly induced the G0/G1 cell cycle arrest in the cells. These data demonstrate that HBCP is a new potent Ca2+/CaM antagonist and can be applied for CaM related therapeutic uses

  12. Structural Properties of Human CaMKII Ca2+ /Calmodulin-Dependent Protein Kinase II using X-ray Crystallography

    Science.gov (United States)

    Cao, Yumeng Melody; McSpadden, Ethan; Kuriyan, John; Department of Molecular; Cell Biology; Department of Chemistry Team

    To this day, human memory storage remains a mystery as we can at most describe the process vaguely on a cellular level. Switch-like properties of Calcium/Calmodulin-Dependent Protein Kinase II make it a leading candidate in understanding the molecular basis of human memory. The protein crystal was placed in the beam of a synchrotron source and the x-ray crystallography data was collected as reflections on a diffraction pattern that undergo Fourier transform to obtain the electron density. We observed two drastic differences from our solved structure at 2.75Å to a similar construct of the mouse CaMKII association domain. Firstly, our structure is a 6-fold symmetric dodecamer, whereas the previously published construct was a 7-fold symmetric tetradecamer. This suggests the association domain of human CaMKII is a dynamic structure that is triggered subunit exchange process. Secondly, in our structure the N-terminal tag is docked as an additional beta-strand on an uncapped beta-sheet present in each association domain protomer. This is concrete evidence of the involvement of the polypeptide docking site in the molecular mechanism underlining subunit exchange. In the future, we would like to selectively inhibit the exchange process while not disrupting the other functionalities of CaMKII.

  13. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    2015-07-01

    Full Text Available Bacillus anthracis adenylyl cyclase toxin edema factor (EF is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH-oxidase, thus reducing production of reactive oxygen species (ROS used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut with Met to leucine (Leu substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

  14. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.

    Science.gov (United States)

    Drmota Prebil, Sara; Slapšak, Urška; Pavšič, Miha; Ilc, Gregor; Puž, Vid; de Almeida Ribeiro, Euripedes; Anrather, Dorothea; Hartl, Markus; Backman, Lars; Plavec, Janez; Lenarčič, Brigita; Djinović-Carugo, Kristina

    2016-01-01

    The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin. PMID:27272015

  15. Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts.

    Science.gov (United States)

    Martin, Tamara P; Lawan, Ahmed; Robinson, Emma; Grieve, David J; Plevin, Robin; Paul, Andrew; Currie, Susan

    2014-02-01

    Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts. PMID:23881186

  16. Intrathecal inhibition of calcium/calmodulin-dependent protein kinase II in diabetic neuropathy adversely affects pain-related behavior.

    Science.gov (United States)

    Jelicic Kadic, Antonia; Boric, Matija; Ferhatovic, Lejla; Banozic, Adriana; Sapunar, Damir; Puljak, Livia

    2013-10-25

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy. PMID:24035897

  17. Effects of Rare Earth Ions on the Interaction between Calmodulin and Melittin%稀土离子对钙调蛋白与蜂毒素作用的影响

    Institute of Scientific and Technical Information of China (English)

    李伟国; 张杰; 赵大庆; 倪嘉缵

    2001-01-01

    The effects of terbium ion on the conformation of calmodulin and on the interaction between calmodulin and melittin have been studied by the endogenous fluorescent spectrometry of calmodulin and melittin,and the sensitized fluorescent spectrometry of terbium ion,respectively.The results show that terbium ions have a tight binding site in the I and II metal-binding sites of calmodulin.The conformation of calmodulin induced by terbium ion can bind melittin and transfer the tryptophane residue of melittin to a relatively hydrophobic environment,while the binding of melittin to calmodulin produces effect on the binding orders of terbium ion in camodulin.Results from FT-IR spectrometry have revealed that upon binding of lanthanum ion,apo-calmodulin undergoes a conformational change with the increase of α-helix content and the decrease of β-sheet content.Melittin's binding to calmodulin has no effect on its conformation induced by the binding of lanthanum ion to calmodulin.%分别用钙调蛋白和蜂毒素的内源荧光光谱以及铽离子的敏化荧光光谱考察了铽离子对钙调蛋白构象变化以及对钙调蛋白与蜂毒素相互作用的影响.结果表明,铽离子首先结合在钙调蛋白的第Ⅰ和第Ⅱ位点,铽离子不影响钙调蛋白与蜂毒素的相互作用,蜂毒素与钙调蛋白作用后不影响铽离子在钙调蛋白上的键合顺序.傅里叶变换红外光谱结果表明三价的镧离子与钙调蛋白作用使钙调蛋白的α螺旋结构增加,β折叠结构减少,与钙离子对它的二级结构影响相类似.稀土离子在钙调蛋白-蜂毒素复合体系中主要与钙调蛋白作用.

  18. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

  19. Curcumin specifically binds to the human calcium-calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies.

    Science.gov (United States)

    Hoda, Nasimul; Naz, Huma; Jameel, Ehtesham; Shandilya, Ashutosh; Dey, Sharmistha; Hassan, Md Imtaiyaz; Ahmad, Faizan; Jayaram, B

    2016-03-01

    Calcium-calmodulin-dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus, it is involved in varieties of cellular functions such as cell signaling and neuronal survival. On the other hand, curcumin, a naturally occurring yellow bioactive component of turmeric possesses wide spectrum of biological actions, and it is widely used to treat atherosclerosis, diabetes, cancer, and inflammation. It also acts as an antioxidant. Here, we studied the interaction of curcumin with human CAMK4 at pH 7.4 using molecular docking, molecular dynamics (MD) simulations, fluorescence binding, and surface plasmon resonance (SPR) methods. We performed MD simulations for both neutral and anionic forms of CAMK4-curcumin complexes for a reasonably long time (150 ns) to see the overall stability of the protein-ligand complex. Molecular docking studies revealed that the curcumin binds in the large hydrophobic cavity of kinase domain of CAMK4 through several hydrophobic and hydrogen-bonded interactions. Additionally, MD simulations studies contributed in understanding the stability of protein-ligand complex system in aqueous solution and conformational changes in the CAMK4 upon binding of curcumin. A significant increase in the fluorescence intensity at 495 nm was observed (λexc = 425 nm), suggesting a strong interaction of curcumin to the CAMK4. A high binding affinity (KD = 3.7 × 10(-8) ± .03 M) of curcumin for the CAMK4 was measured by SPR further indicating curcumin as a potential ligand for the CAMK4. This study will provide insights into designing a new inspired curcumin derivatives as therapeutic agents against many life-threatening diseases. PMID:25929263

  20. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    Science.gov (United States)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  1. Abscisic acid activates a Ca2+-calmodulin-stimulated protein kinase involved in antioxidant defense in maize leaves

    Institute of Scientific and Technical Information of China (English)

    Shucheng Xu

    2010-01-01

     The role of a calcium-dependent and calmodulin(CaM)stimulated protein kinase in abscisic acid(ABA)-induced antioxidant defense was determined in leaves of maize (Zea mays).In-gel kinase assays showed that treatments with ABA or H2O2 induced the activation of a 49-kDa protein kinase and a 52-kDa protein kinase significantly.Furthermore,we showed that the 52-kDa protein kinase has the characteristics of CaM-stimulating activity and is sensitive to calcium-CaM-dependent protein kinase Ⅱ (CaMK Ⅱ)inhibitor KN-93 or CaM antagonist W-7.Treatments with ABA or H2O2 not only induced the acti vation of the 52-kDa protein kinase,but also enhanced the total activities of the antioxidant enzymes,including catalase,ascorbate peroxidase,glutathione reductase,and superoxide dismutase.Such enhancements were blocked by pretreatment with a CaMK inhibitor and a reactive oxygen species(ROS)inhibitor or scavenger.Pretreatment with the CaMK inhibitor also substantially arrested the ABA-induced H2O2 production.Kinase activity enhancements induced by ABA were attenuated by pretreatment with an ROS inhibitor or scavenger.These results suggest that the 52-kDa CaMK is involved in ABA-induced antioxidant defense and that cross-talk between CaMK and H2O2 plays a pivotal role in ABA signaling.We infer that CaMK acts both upstream and downstream of H2O2,but mainly acts between ABA and H2O2 in ABA-induced antioxidant-defensive signaling.

  2. Regulation of plant immunity through ubiquitin-mediated modulation of Ca(2+) -calmodulin-AtSR1/CAMTA3 signaling.

    Science.gov (United States)

    Zhang, Lei; Du, Liqun; Shen, Chenjia; Yang, Yanjun; Poovaiah, B W

    2014-04-01

    Transient changes in intracellular Ca(2+) concentration are essential signals for activation of plant immunity. It has also been reported that Ca(2+) signals suppress salicylic acid-mediated plant defense through AtSR1/CAMTA3, a member of the Ca(2+) /calmodulin-regulated transcription factor family that is conserved in multicellular eukaryotes. How plants overcome this negative regulation to mount an effective defense response during a stage of intracellular Ca(2+) surge is unclear. Here we report the identification and functional characterization of an important component of ubiquitin ligase, and the associated AtSR1 turnover. The AtSR1 interaction protein 1 (SR1IP1) was identified by CytoTrap two-hybrid screening. The loss-of-function mutant of SR1IP1 is more susceptible to bacterial pathogens, and over-expression of SR1IP1 confers enhanced resistance, indicating that SR1IP1 acts as a positive regulator of plant defense. SR1IP1 and AtSR1 act in the same signaling pathway to regulate plant immunity. SR1IP1 contains the structural features of a substrate adaptor in cullin 3-based E3 ubiquitin ligase, and was shown to serve as a substrate adaptor that recruits AtSR1 for ubiquitination and degradation when plants are challenged with pathogens. Hence, SR1IP1 positively regulates plant immunity by removing the defense suppressor AtSR1. These findings provide a mechanistic insight into how Ca(2+) -mediated actions are coordinated to achieve effective plant immunity. PMID:24528504

  3. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    International Nuclear Information System (INIS)

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca2+ and this interaction may be important for its invasion into animal cells

  4. Novel regulation of equlibrative nucleoside transporter 1 (ENT1) by receptor-stimulated Ca2+-dependent calmodulin binding.

    Science.gov (United States)

    Bicket, Alex; Mehrabi, Pedram; Naydenova, Zlatina; Wong, Victoria; Donaldson, Logan; Stagljar, Igor; Coe, Imogen R

    2016-05-15

    Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (∼12% and ∼39%, respectively) by chelating calcium (EGTA, 50 μM; BAPTA-AM, 25 μM), whereas increasing intracellular calcium (thapsigargin, 1.5 μM) led to increased nucleoside uptake (∼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 μM) significantly increased nucleoside uptake (∼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 μM), or CaM antagonist, W7 (50 μM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy. PMID:27009875

  5. Nicotine reward and affective nicotine withdrawal signs are attenuated in calcium/calmodulin-dependent protein kinase IV knockout mice.

    Directory of Open Access Journals (Sweden)

    Kia J Jackson

    Full Text Available The influx of Ca(2+ through calcium-permeable nicotinic acetylcholine receptors (nAChRs leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB, which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (-/- mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV -/- mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.

  6. Mycorrhizal-induced calmodulin mediated changes in antioxidant enzymes and growth response of drought-stressed trifoliate orange

    Directory of Open Access Journals (Sweden)

    Yong-Ming eHuang

    2014-12-01

    Full Text Available Trifoliate orange [Poncirus trifoliata (L Raf.] is considered highly arbuscular mycorrhizal (AM dependent for growth responses through a series of signal transductions in form of various physiological responses. The proposed study was carried out to evaluate the effect of an AM fungus (Funneliformis mosseae on growth, antioxidant enzyme (catalase, CAT; superoxide dismutase, SOD activities, leaf relative water content (RWC, calmodulin (CaM, superoxide anion (O2•− and hydrogen peroxide (H2O2 concentrations in leaves of the plants exposed to both well-watered (WW and drought stress (DS conditions. A 58-day of DS significantly decreased mycorrhizal colonization by 60% than WW. Compared to non-AM seedlings, AM seedlings displayed significantly higher shoot morphological properties (plant height, stem diameter and leaf number, biomass production (shoot and root fresh weight and leaf RWC, regardless of soil water status. AM inoculation significantly increased CaM and soluble protein concentrations and CAT activity, and significantly decreased O2•− and H2O2 concentration under both WW and DS conditions. The AM seedlings also exhibited significantly higher Cu/Zn-SOD and Mn-SOD activities than the non-AM seedlings under DS but not under WW, which are triggered by higher CaM levels in AM plants on the basis of correlation studies. Further, the negative correlation of Cu/Zn-SOD and Mn-SOD activities with O2•− and H2O2 concentration showed the DS-induced ROS scavenging ability of CaM mediated SODs under mycorrhization. Our results demonstrated that AM-inoculation elevated the synthesis of CaM in leaves and up-regulated activities of the antioxidant enzymes, thereby, repairing the possible oxidative damage to plants by lowering the ROS accumulation under DS condition.

  7. Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System.

    Science.gov (United States)

    Ishida, Hiroaki; Nguyen, Leonard T; Gopal, Ramamourthy; Aizawa, Tomoyasu; Vogel, Hans J

    2016-09-01

    In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells. PMID:27502305

  8. Molecular characterisation of a calmodulin gene, VcCaM1, that is differentially expressed under aluminium stress in highbush blueberry.

    Science.gov (United States)

    Inostroza-Blancheteau, C; Aquea, F; Loyola, R; Slovin, J; Josway, S; Rengel, Z; Reyes-Díaz, M; Alberdi, M; Arce-Johnson, P

    2013-11-01

    Calmodulin (CaM), a small acidic protein, is one of the best characterised Ca(2+) sensors in eukaryotes. This Ca(2+) -regulated protein plays a critical role in decoding and transducing environmental stress signals by activating specific targets. Many environmental stresses elicit changes in intracellular Ca(2+) activity that could initiate adaptive responses under adverse conditions. We report the first molecular cloning and characterisation of a calmodulin gene, VcCaM1 (Vaccinium corymbosum Calmodulin 1), in the woody shrub, highbush blueberry. VcCaM1 was first identified as VCAL19, a gene induced by aluminium stress in V. corymbosum L. A full-length cDNA of VcCaM1 containing a 766-bp open reading frame (ORF) encoding 149 amino acids was cloned from root RNA. The sequence encodes four Ca(2+) -binding motifs (EF-hands) and shows high similarity (99%) with the isoform CaM 201 of Daucus carota. Expression analyses showed that following Al treatment, VcCaM1 message level decreased in roots of Brigitta, an Al-resistant cultivar, and after 48 h, was lower than in Bluegold, an Al-sensitive cultivar. VcCAM1 message also decreased in leaves of both cultivars within 2 h of treatment. Message levels in leaves then increased by 24 h to control levels in Brigitta, but not in Bluegold, but then decreased again by 48 h. In conclusion, VcCaM1 does not appear to be directly involved in Al resistance, but may be involved in improved plant performance under Al toxicity conditions through regulation of Ca(2+) homeostasis and antioxidant systems in leaves. PMID:23627459

  9. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongliang; Hong, Da Hye; Kim, Han Sol; Kim, Hye Won [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of); Jung, Won-Kyo [Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan 608-737 (Korea, Republic of); Na, Sung Hun [Institute of Medical Sciences, Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, 200-701 (Korea, Republic of); Jung, In Duk; Park, Yeong-Min [Department of Immunology, Lab of Dendritic Cell Differentiation and Regulation, College of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Choi, Il-Whan, E-mail: cihima@inje.ac.kr [Department of Microbiology, Inje University College of Medicine, Busan, 614-735 (Korea, Republic of); Park, Won Sun, E-mail: parkws@kangwon.ac.kr [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of)

    2015-06-15

    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

  10. Calmodulin 2 Mutation N98S Is Associated with Unexplained Cardiac Arrest in Infants Due to Low Clinical Penetrance Electrical Disorders

    Science.gov (United States)

    Jiménez-Jáimez, Juan; Palomino Doza, Julián; Ortega, Ángeles; Macías-Ruiz, Rosa; Perin, Francesca; Rodríguez-Vázquez del Rey, M. Mar; Ortiz-Genga, Martín; Monserrat, Lorenzo; Barriales-Villa, Roberto; Blanca, Enrique; Álvarez, Miguel; Tercedor, Luis

    2016-01-01

    Background Calmodulin 1, 2 and 3 (CALM) mutations have been found to cause cardiac arrest in children at a very early age. The underlying aetiology described is long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT) and idiopathic ventricular fibrillation (IVF). Little phenotypical data about CALM2 mutations is available. Objectives The aim of this paper is to describe the clinical manifestations of the Asn98Ser mutation in CALM2 in two unrelated children in southern Spain with apparently unexplained cardiac arrest/death. Methods Two unrelated children aged 4 and 7, who were born to healthy parents, were studied. Both presented with sudden cardiac arrest. The first was resuscitated after a VF episode, and the second died suddenly. In both cases the baseline QTc interval was within normal limits. Peripheral blood DNA was available to perform targeted gene sequencing. Results The surviving 4-year-old girl had a positive epinephrine test for LQTS, and polymorphic ventricular ectopic beats were seen on a previous 24-hour Holter recording from the deceased 7-year-old boy, suggestive of a possible underlying CPVT phenotype. A p.Asn98Ser mutation in CALM2 was detected in both cases. This affected a highly conserved across species residue, and the location in the protein was adjacent to critical calcium binding loops in the calmodulin carboxyl-terminal domain, predicting a high pathogenic effect. Conclusions Human calmodulin 2 mutation p.Asn98Ser is associated with sudden cardiac death in childhood with a variable clinical penetrance. Our results provide new phenotypical information about clinical behaviour of this mutation. PMID:27100291

  11. Targeting cell migration and the endoplasmic reticulum stress response with calmodulin antagonists: a clinically tested small molecule phenocopy of SEC62 gene silencing in human tumor cells

    International Nuclear Information System (INIS)

    Tumor cells benefit from their ability to avoid apoptosis and invade other tissues. The endoplasmic reticulum (ER) membrane protein Sec62 is a key player in these processes. Sec62 is essential for cell migration and protects tumor cells against thapsigargin-induced ER stress, which are both linked to cytosolic Ca2+. SEC62 silencing leads to elevated cytosolic Ca2+ and increased ER Ca2+ leakage after thapsigargin treatment. Sec62 protein levels are significantly increased in different tumors, including prostate, lung and thyroid cancer. In lung cancer, the influence of Sec62 protein levels on patient survival was analyzed using the Kaplan-Meier method and log-rank test. To elucidate the underlying pathophysiological functions of Sec62, Ca2+ imaging techniques, real-time cell analysis and cell migration assays were performed. The effects of treatment with the calmodulin antagonists, trifluoperazine (TFP) and ophiobolin A, on cellular Ca2+ homeostasis, cell growth and cell migration were compared with the effects of siRNA-mediated Sec62 depletion or the expression of a mutated SEC62 variant in vitro. Using Biacore analysis we examined the Ca2+-sensitive interaction of Sec62 with the Sec61 complex. Sec62 overproduction significantly correlated with reduced patient survival. Therefore, Sec62 is not only a predictive marker for this type of tumor, but also an interesting therapeutic target. The present study suggests a regulatory function for Sec62 in the major Ca2+ leakage channel in the ER, Sec61, by a direct and Ca2+-sensitive interaction. A Ca2+-binding motif in Sec62 is essential for its molecular function. Treatment of cells with calmodulin antagonists mimicked Sec62 depletion by inhibiting cell migration and rendering the cells sensitive to thapsigargin treatment. Targeting tumors that overproduce Sec62 with calmodulin antagonists in combination with targeted thapsigargin analogues may offer novel personalized therapeutic options

  12. Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

    Science.gov (United States)

    Lou, Jie; Zhao, Dan; Zhang, Ling-Ling; Song, Shu-Ying; Li, Yan-Chao; Sun, Fei; Ding, Xiao-Qing; Yu, Chang-Jiang; Li, Yuan-Yuan; Liu, Mei-Tong; Dong, Chang-Jiang; Ji, Yong; Li, Hongliang; Chu, Wenfeng; Zhang, Zhi-Ren

    2016-09-01

    The role of type III transforming growth factor-β receptor (TβRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TβRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TβRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TβRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TβRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TβRIII in vitro. Cardiac-specific transgenic expression of TβRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TβRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TβRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TβRIII in vivo. Our data suggest that TβRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of β-arrestin2 with both TβRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TβRIII expression results in cardiac hypertrophy through β-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-β and β-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac

  13. Ca2+ Binding Protein S100A1 Competes with Calmodulin and PIP2 for Binding Site on the C-Terminus of the TPRV1 Receptor

    Czech Academy of Sciences Publication Activity Database

    Gryčová, Lenka; Holendová, Blanka; Lánský, Zdeněk; Bumba, Ladislav; Jirků, Michaela; Boušová, Kristýna; Teisinger, Jan

    2015-01-01

    Roč. 6, č. 3 (2015), s. 386-392. ISSN 1948-7193 R&D Projects: GA ČR(CZ) GAP301/10/1159; GA ČR(CZ) GAP207/11/0717; GA ČR(CZ) GA15-11851S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 ; RVO:67985823 ; RVO:61388971 Keywords : TRPV1 * vanilloid receptor * calmodulin * S100A1 * calcium binding protein * surface plasmon resonance * fluorescence anisotropy Subject RIV: CE - Biochemistry Impact factor: 4.362, year: 2014

  14. Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

    OpenAIRE

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M.

    2009-01-01

    Voltage-activated Cav1.2 calcium channels require association of the pore-forming α1C subunit with accessory Cavβ and α2δ subunits. Binding of a single calmodulin (CaM) to α1C supports Ca2+-dependent inactivation (CDI). The human Cav1.2 channel is silent in the absence of Cavβ and/or α2δ. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Cavβ. Here we discovered that CaMex and its Ca2+...

  15. Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: High-resolution mechanistic implications for channel regulation by Ca2+

    OpenAIRE

    Mori, Masayuki X.; Vander Kooi, Craig W.; Leahy, Daniel J.; Yue, David T.

    2008-01-01

    Calmodulin (CaM) regulation of Ca2+ channels is central to Ca2+ signaling. CaV1 versus CaV2 classes of these channels exhibit divergent forms of regulation, potentially relating to customized CaM/IQ interactions among different channels. Here, we report the crystal structures for the Ca2+/CaM—IQ domains of both CaV2.1 and CaV2.3 channels. These highly similar structures emphasize that major CaM contacts with the IQ domain extend well upstream of traditional consensus residues. Surprisingly, u...

  16. A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed.

    OpenAIRE

    Martin, J R; Ollo, R

    1996-01-01

    Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. We have cloned a new Drosophila CaM kinase gene named caki. We describe the molecular characterization of caki and a behavioral effect of its elimination. The caki gene is extremely large; comparison of the genomic and cDNA sequences reveals that the caki transcription unit is at least 150 kb. The catalytic domain of this new CaM kinase protein shares homology (41%) with type II Ca...

  17. Ca2+/Calmodulin Kinase Kinase α Is Dispensable for Brain Development but Is Required for Distinct Memories in Male, though Not in Female, Mice▿

    OpenAIRE

    Mizuno, Keiko; Ris, Laurence; Sánchez-Capelo, Amelia; Godaux, Emile; Giese, K. Peter

    2006-01-01

    In neurons, the Ca2+/calmodulin (CaM) kinase cascade transduces Ca2+ signaling into gene transcription. The CaM kinase cascade is known to be important for brain development as well as memory formation in adult brain, although the functions of some cascade members remain unknown. Here we have generated null and hypomorphic mutants to study the physiological role of CaM kinase kinase α (CaMKKα), which phosphorylates and activates both CaM kinase I (CaMKI) and CaMKIV, the output kinases of the ...

  18. Regulation of Voltage-Gated Ca2+ Currents by Ca2+/Calmodulin-dependent Protein Kinase II in Resting Sensory Neurons

    OpenAIRE

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-en; Hogan, Quinn H

    2014-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca2+ channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca2+ currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced...

  19. Is buffer a good proxy for a crowded cell-like environment? A comparative NMR study of calmodulin side-chain dynamics in buffer and E. coli lysate.

    Directory of Open Access Journals (Sweden)

    Michael P Latham

    Full Text Available Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded "cell-like" environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca(2+-bound and Ca(2+, peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed "test-tube" studies, experiments performed under conditions that are "cell-like" are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

  20. A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin.

    Science.gov (United States)

    Lawrence, Anthony J; Coote, John G; Kazi, Yasmin F; Lawrence, Paul D; MacDonald-Fyall, Julia; Orr, Barbara M; Parton, Roger; Riehle, Mathis; Sinclair, James; Young, John; Price, Nicholas C

    2002-06-21

    Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated. PMID:11934879

  1. Overexpression of calmodulin-like (ShCML44) stress-responsive gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses.

    Science.gov (United States)

    Munir, Shoaib; Liu, Hui; Xing, Yali; Hussain, Saddam; Ouyang, Bo; Zhang, Yuyang; Li, Hanxia; Ye, Zhibiao

    2016-01-01

    Calmodulin-like (CML) proteins are important Ca(2+) sensors, which play significant role in mediating plant stress tolerance. In the present study, cold responsive calmodulin-like (ShCML44) gene was isolated from cold tolerant wild tomato (Solanum habrochaites), and functionally characterized. The ShCML44 was differentially expressed in all plant tissues including root, stem, leaf, flower and fruit, and was strongly up-regulated under cold, drought and salinity stresses along with plant growth hormones. Under cold stress, progressive increase in the expression of ShCML44 was observed particularly in cold-tolerant S. habrochaites. The ShCML44-overexpressed plants showed greater tolerance to cold, drought, and salinity stresses, and recorded higher germination and better seedling growth. Transgenic tomato plants demonstrated higher antioxidant enzymes activity, gas exchange and water retention capacity with lower malondialdehyde accumulation and membrane damage under cold and drought stresses compared to wild-type. Moreover, transgenic plants exhibited reduced reactive oxygen species and higher relative water contents under cold and drought stress, respectively. Greater stress tolerance of transgenic plants was further reflected by the up-/down-regulation of stress-related genes including SOD, GST, CAT, POD, LOX, PR and ERD. In crux, these results strengthen the molecular understanding of ShCML44 gene to improve the abiotic stress tolerance in tomato. PMID:27546315

  2. The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

    Science.gov (United States)

    Kool, Martijn J; van de Bree, Jolet E; Bodde, Hanna E; Elgersma, Ype; van Woerden, Geeske M

    2016-01-01

    Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2b(f/f) and Camk2b(T287A)), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b(-/-) mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion. PMID:27244486

  3. The solution structures of two soybean calmodulin isoforms provide a structural basis for their selective target activation properties.

    Science.gov (United States)

    Ishida, Hiroaki; Huang, Hao; Yamniuk, Aaron P; Takaya, Yoshiaki; Vogel, Hans J

    2008-05-23

    The intracellular calcium ion is one of the most important secondary messengers in eukaryotic cells. Ca(2+) signals are translated into physiological responses by EF-hand calcium-binding proteins such as calmodulin (CaM). Multiple CaM isoforms occur in plant cells, whereas only a single CaM protein is found in animals. Soybean CaM isoform 1 (sCaM1) shares 90% amino acid sequence identity with animal CaM (aCaM), whereas sCaM4 is only 78% identical. These two sCaM isoforms have distinct target-enzyme activation properties and physiological functions. sCaM4 is highly expressed during the self-defense reaction of the plant and activates the enzyme nitric-oxide synthase (NOS), whereas sCaM1 is incapable of activating NOS. The mechanism of selective target activation by plant CaM isoforms is poorly understood. We have determined high resolution NMR solution structures of Ca(2+)-sCaM1 and -sCaM4. These were compared with previously determined Ca(2+)-aCaM structures. For the N-lobe of the protein, the solution structures of Ca(2+)-sCaM1, -sCaM4, and -aCaM all closely resemble each other. However, despite the high sequence identity with aCaM, the C-lobe of Ca(2+)-sCaM1 has a more open conformation and consequently a larger hydrophobic target-protein binding pocket than Ca(2+)-aCaM or -sCaM4, the presence of which was further confirmed through biophysical measurements. The single Val-144 --> Met substitution in the C-lobe of Ca(2+)-sCaM1, which restores its ability to activate NOS, alters the structure of the C-lobe to a more closed conformation resembling Ca(2+)-aCaM and -sCaM4. The relationships between the structural differences in the two Ca(2+)-sCaM isoforms and their selective target activation properties are discussed. PMID:18347016

  4. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  5. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93 calmodulin cDNA using computational tools

    Directory of Open Access Journals (Sweden)

    Kassim Amelia

    2015-01-01

    Full Text Available Background: Common bean (Phaseolus vulgaris L. is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM gene cDNA and its deduced protein (amino acids sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is

  6. An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility.

    Science.gov (United States)

    Suarez, Andres; Huber, Robert J; Myre, Michael A; O'Day, Danton H

    2011-07-01

    CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote. PMID:21402150

  7. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools

    Science.gov (United States)

    Amelia, Kassim; Singh, Jasvin; Shah, Farida Habib; Bhore, Subhash J.

    2015-01-01

    Background: Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day and 20-day old bean-pod-tissues. Altogether, 5972 cDNA clones were isolated to have ESTs. While processing ESTs, we found a transcript for calmodulin (CaM) gene. It is an important gene that encodes for a calcium-binding protein and known to express in all eukaryotic cells. Hence, this study was undertaken to analyse and annotate it. Objective: The objective of this study was to analyze and annotate P. vulgaris CaM (PvCaM) gene cDNA and its deduced protein (amino acids) sequence. Materials and Methods: Both strands of PvCaM cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. The cDNA sequence and deduced protein sequence were analyzed and annotated using bioinformatics tools available online. The secondary structures and three-dimensional (3D) structure of PvCaM protein were predicted using the Phyre automatic fold recognition server. Results: Results showed that PvCaM cDNA is 818 bp in length. The cDNA analysis results showed that it contains an open reading frame that encodes for 149 amino acid residues. The deduced protein sequence analysis results showed the presence of conserved domains required for CaM function. The predicted secondary structures and 3D structure are analogous to the Solanum tuberosum CaM. Conclusions: This study analyzed and annotated PvCaM cDNA and protein. However, in order to obtain a complete understanding of PvCaM protein, further study on its expression, structure and regulation is essential. PMID

  8. A many-body model to study proteins. II. Incidence of many-body polarization effects on the interaction of the calmodulin protein with four Ca2+ dications and with a target enzyme peptide

    Science.gov (United States)

    Cuniasse, Philippe; Masella, Michel

    2003-07-01

    The origin of the interactions occurring in the calmodulin protein interacting with one of its target peptide and counterions, and binding four calcium dications, has been investigated in the gas phase, using the many-body model presented in Paper I [Masella and Cuniasse, J. Chem. Phys. 119, 1866 (2003)] and a classical pairwise force field. As compared to the latter force field, the many-body model is shown to provide a geometrical description of the calmodulin/target peptide structure in better agreement with the x-ray experimental one, and a better description of the Ca2+ binding sites (as compared to "small molecule" structures reported in the Cambridge Structural Database). Regarding the energy, both models provide qualitatively a similar description of the interactions occurring in the calmodulin/target peptide system. However, quantitatively, the pairwise model predicts interaction energies greater by about 25% as compared to the many-body one in the case of calmodulin/Ca2+ interactions. This is due to the inability of pairwise force fields to account for the strong anticooperative effects predicted to occur in [Ca,(carboxylate)n]2-n systems by both the many-body model and quantum computations. Hence, the new many-body model appears to be well suited for describing proteinic systems interacting with cations, both in terms of geometry and energy.

  9. Small-angle x-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly. alpha. -helical conformation

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Mikio; Engelman, D.M. (Yale Univ., New Haven, CT (USA)); Head, J.F. (Boston Univ., MA (USA)); Persechini, A.; Kretsinger, R.H. (Univ. of Virginia, Charlottesville (USA))

    1991-02-05

    Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca{sup 2+}, the observed radii of gyration are 22.4 {angstrom} for wild-type bacterially expressed calmodulin, 19.5 {angstrom} for Des2 calmodulin, and 20.3 {angstrom} for Des4 calmodulin. A reduction in the radius of gyration by 1-2 {angstrom} on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows that the structural properties of the deletion mutants are well described by modifications of an {alpha} helix in the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.

  10. Small-angle x-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly α-helical conformation

    International Nuclear Information System (INIS)

    Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 angstrom for wild-type bacterially expressed calmodulin, 19.5 angstrom for Des2 calmodulin, and 20.3 angstrom for Des4 calmodulin. A reduction in the radius of gyration by 1-2 angstrom on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows that the structural properties of the deletion mutants are well described by modifications of an α helix in the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide

  11. Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: an RNAi and yeast two-hybrid study

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    Gonzalez-Mendez Ricardo

    2011-07-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1 gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yeast cells as would be expected. The level of sscmk1 gene expression in RNAi transformants at 35°C was less than that of cells transformed with the empty pSD2G at this same temperature. Yeast two-hybrid analysis of proteins that interact with SSCMK1 identified a homologue of heat shock protein 90 (HSP90 as interacting with this kinase. Growth of the fungus similar to that of the RNAi transformants was observed in medium with geldanamycin (GdA, 10 μM, an inhibitor of HSP90. Conclusions Using the RNAi technology we silenced the expression of sscmk1 gene in this fungus. RNAi transformants were unable to grow as yeast cells at 35°C showing decreased tolerance to this temperature. The interaction of SSCMK1 with HSP90, observed using the yeast two-hybrid assay suggests that this kinase is involved in thermotolerance through its interaction with HSP90. SSCMK1 interacted with the C terminal domain of HSP90 where effector proteins and co-chaperones interact. These

  12. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

    Energy Technology Data Exchange (ETDEWEB)

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  13. Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: calmodulin dendrograms show significant lack of parallelism

    Science.gov (United States)

    Nakayama, S.; Kretsinger, R. H.

    1993-01-01

    In the first report in this series we presented dendrograms based on 152 individual proteins of the EF-hand family. In the second we used sequences from 228 proteins, containing 835 domains, and showed that eight of the 29 subfamilies are congruent and that the EF-hand domains of the remaining 21 subfamilies have diverse evolutionary histories. In this study we have computed dendrograms within and among the EF-hand subfamilies using the encoding DNA sequences. In most instances the dendrograms based on protein and on DNA sequences are very similar. Significant differences between protein and DNA trees for calmodulin remain unexplained. In our fourth report we evaluate the sequences and the distribution of introns within the EF-hand family and conclude that exon shuffling did not play a significant role in its evolution.

  14. MICrocephaly, disproportionate pontine and cerebellar hypoplasia syndrome: A clinico-radiologic phenotype linked to calcium/calmodulin-dependent serine protein kinase gene mutation

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    Rashid Saleem

    2013-01-01

    Full Text Available MICrocephaly, disproportionate pontine and cerebellar hypoplasia (MICPCH syndrome, a rare X-linked disorder, generally seen in girls, is characterized by neurodevelopmental delay, microcephaly, and disproportionate pontine and cerebellar hypoplasia. It is caused by inactivating calcium/calmodulin-dependent serine protein kinase (CASK gene mutations. We report a 2-year-old girl with severe neurodevelopmental delay, microcephaly, minimal pontine hypoplasia, cerebellar hypoplasia, and normal looking corpus callosum, with whom the conventional cytogenetic studies turned out to be normal, and an array-comparative genomic hybridization (a-CGH analysis showed CASK gene duplication at Xp11.4. Our case highlights the importance of using clinico-radiologic phenotype to guide genetic investigation and it also confirms the role of a-CGH analysis in establishing the genetic diagnosis of MICPCH syndrome, when conventional cytogenetic studies are inconclusive.

  15. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    Science.gov (United States)

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  16. Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD.

    Science.gov (United States)

    Lambert, Charles C

    2011-01-01

    Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis. PMID:21774024

  17. Digoxin and ouabain induce P-glycoprotein by activating calmodulin kinase II and hypoxia-inducible factor-1α in human colon cancer cells

    International Nuclear Information System (INIS)

    Digoxin and ouabain are cardioactive glycosides, which inhibit the Na+/K+-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca++]i). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca++]i and this event was dependent on the calcium influx via the Na+/Ca++ exchanger. The increased [Ca++]i enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1α. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na+/Ca++ exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

  18. Phylogeny of plant calcium and calmodulin-dependent protein kinases (CCaMKs and functional analyses of tomato CCaMK in disease resistance

    Directory of Open Access Journals (Sweden)

    Ji-Peng eWang

    2015-12-01

    Full Text Available Calcium and calmodulin-dependent protein kinase (CCaMK is a member of calcium/calmodulin-dependent protein kinase superfamily and is essential to microbe- plant symbiosis. To date, the distribution of CCaMK gene in plants has not yet been completely understood, and its function in plant disease resistance remains unclear. In this study, we systemically identified the CCaMK genes in genomes of 44 plant species in Phytozome and analyzed the function of tomato CCaMK (SlCCaMK in resistance to various pathogens. CCaMKs in 18 additional plant species were identified, yet the absence of CCaMK gene in green algae and cruciferous species was confirmed. Sequence analysis of full-length CCaMK proteins from 44 plant species demonstrated that plant CCaMKs are highly conserved across all domains. Most of the important regulatory amino acids are conserved throughout all sequences, with the only notable exception being observed in N-terminal autophosphorylation site corresponding to Ser 9 in the Medicago truncatula CCaMK. CCaMK gene structures are similar, mostly containing six introns with a phase profile of 200200 and the exception was only noticed at the first exons. Phylogenetic analysis demonstrated that CCaMK lineage is likely to have diverged early from a calcium-dependent protein kinase (CDPK gene in the ancestor of all nonvascular plant species. The SlCCaMK gene was widely and differently responsive to diverse pathogenic stimuli. Furthermore, knock-down of SlCCaMK reduced tomato resistance to Sclerotinia sclerotiorum and Pseudomonas syringae pv. tomato (Pst DC3000 and decreased H2O2 accumulation in response to Pst DC3000 inoculation. Our results reveal that SlCCaMK positively regulates disease resistance in tomato via promoting H2O2 accumulation. SlCCaMK is the first CCaMK gene proved to function in plant disease resistance.

  19. Dendritic spine changes in the development of alcohol addiction regulated by α-calcium/calmodulin-dependent protein kinase II

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    Zofia Mijakowska

    2014-03-01

    Full Text Available Introduction Alcohol has many adverse effects on the brain. Among them are dendritic spine morphology alterations, which are believed to be the basis of alcohol addiction. Autophosphorylation of α-calcium/calmodulin-dependent protein kinase II (αCaMKII has been shown to regulate spine morphology in vitro. Here we show that αCaMKII can also regulate addiction related behaviour and dendritic spine morphology changes caused by alcohol consumption in vivo. Method 12 αCaMKII-autophosphorylation deficient female mice (T286A and 12 wild type littermates were used in the study. T286A strain was created by Giese et al. (1998. Mice were housed and tested in two IntelliCages from NewBehavior (www.newbehavior.com. IntelliCage is an automated learning system. After 95 days of alcohol drinking interrupted by tests for motivation, persistence in alcohol seeking and probability of relapse, mice were ascribed to ‘high’ or ‘low’ drinkers group according to their performance in the tests. Additional criterion was the amount of alcohol consumed during the whole experiment. Result of each test was evaluated separately. 1/3 of the mice that scored highest in each criterion were considered ‘positive’ for this trait. ‘Positive’ animals were given 1 point, negative 0 points. Mice that were positive in at least 2 criteria were ascribed to ‘high’ drinkers (‘+’ group. Remaining mice – to ‘low’ drinkers (‘–‘. This method of behavioral phenotyping, developed by Radwanska and Kaczmarek (2012, is inspired by DSM-IV. Since the results of this evaluation are discrete (i.e. by definition all the animals score between 0 to +4, we developed also a continuous method of addiction rating, which we call ‘addiction index’. The result of the second method is a sum of the standardized (z-score results of the above mentioned tests. We use it to examine the correlations between addiction-like behavior and spine parameters. Control group (12 WT, 8

  20. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells.

    Science.gov (United States)

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  1. Calmodulin of the tropical sea cucumber: Gene structure, inducible expression and contribution to nitric oxide production and pathogen clearance during immune response.

    Science.gov (United States)

    Chen, Ting; Ren, Chunhua; Li, Wuhu; Jiang, Xiao; Xia, Jianjun; Wong, Nai-Kei; Hu, Chaoqun

    2015-08-01

    Calmodulin (CaM) is an essential second messenger protein that transduces calcium signals by binding calcium ions (Ca(2+)) and modulating its interactions with various target proteins. In contrast to vertebrates, where CaM is well established as a cofactor for Ca(2+)-dependent physiological and cellular functions including host defense, there is a paucity of understanding on CaM in invertebrates (such as echinoderms) in response to immune challenge or microbial infections. In this study, we obtained and described the gene sequence of CaM from the tropical sea cucumber Stichopus monotuberculatus, a promising yet poorly characterized aquacultural species. mRNA expression of StmCaM could be detected in the intestine and coelomic fluid after Vibrio alginolyticus injection. Transcriptional and translational expression of StmCaM was inducible in nature, as evidenced by the expression patterns in primary coelomocytes following Vibrio challenge. This response could be mimicked by the Vibrio cells membrane components or lipopolysaccharides (LPS), and blocked by co-treatment of the LPS-neutralizing agent polymyxin B (PMB). Furthermore, inhibition of CaM activity by incubation with its inhibitor trifluoroperazine dihydrochloride (TFP) blunted the production of Vibrio-induced nitric oxide (NO) and augmented the survival of invading Vibrio in coelomocytes. Collectively, our study here supplied the first evidence for echinoderm CaM participation in innate immunity, and provided a functional link between CaM expression and antibacterial NO production in sea cucumber. PMID:25913576

  2. Effects of tiflucarbine as a dual protein kinase C/calmodulin antagonist on proliferation of human keratinocytes and release of reactive oxygen species from human leukocytes.

    Science.gov (United States)

    Hegemann, L; Fruchtmann, R; Bonnekoh, B; Schmidt, B H; Traber, J; Mahrle, G; Müller-Peddinghaus, R; van Rooijen, L A

    1991-01-01

    Various studies have suggested that calmodulin (CaM) is involved in the pathophysiology of psoriasis. Protein kinase C (PKC) is also accepted as playing a regulatory role in cell proliferation as well as in inflammatory processes. Therefore, we investigated the effects of the known CaM antagonist tiflucarbine (BAY/TVX P 4495) on two cellular systems related to the major clinical symptoms of psoriasis: proliferation of cultured human keratinocytes (HaCa T cell line) and release of reactive oxygen species (ROS) from human polymorphonuclear leukocytes (PMNL). Tiflucarbine inhibited both cellular responses in a dose dependent manner. Furthermore, tiflucarbine directly affected PKC, and may thus be considered to be a dual PKC/CaM antagonist with putative antipsoriatic activity. The effects of tiflucarbine on the different parameters were compared with those of the structurally unrelated dual PKC/CaM inhibitor W-7 and those of the potent PKC inhibitor staurosporine. The potencies of all three compounds were found to be in the same range as their PKC-inhibiting potency. Our data indicate that PKC, rather than CaM, may play a regulatory role in the release of ROS as well as in keratinocyte proliferation. Therefore, inhibition of PKC in general might have a therapeutic benefit in psoriasis. PMID:1801655

  3. Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

    Science.gov (United States)

    de Souza, Tatiana de Arruda Campos Brasil; Graça-de Souza, Viviane Krominski; Lancheros, César Armando Contreras; Monteiro-Góes, Viviane; Krieger, Marco Aurélio; Goldenberg, Samuel; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

    2011-03-01

    In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity. PMID:21431874

  4. Involvement of Ca2+/calmodulin-dependent protein kinase II in the activation of carnitine palmitoyltransferase I by okadaic acid in rat hepatocytes.

    Science.gov (United States)

    Velasco, G; Guzmán, M; Zammit, V A; Geelen, M J

    1997-01-01

    The present work was undertaken to study the mechanism by which okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, stimulates carnitine palmitoyltransferase I (CPT-I) in isolated rat hepatocytes [Guzmán, Kolodziej, Caldwell, Costorphine and Zammit (1994) Biochem. J. 300, 693-699]. The OA-induced stimulation of CPT-I was abolished by the general protein kinase inhibitor K-252a as well as by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII). However, neither the protein kinase C-specific inhibitor bisindolylmaleimide nor the protein kinase A/protein kinase C inhibitor H-7 was able to prevent the OA-induced stimulation of CPT-I. Hepatocyte-shrinkage-induced stimulation of CPT-I as well as OA-induced hepatocyte shrinkage was prevented by KN-62. KN-62 also antagonized the OA-enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased the degree of phosphorylation of Ca2+/CM-PKII, as immunoprecipitated by a monoclonal antibody raised against the alpha-subunit of rat brain kinase. This effect of OA was also antagonized by KN-62. The results thus indicate that the OA-dependent stimulation of CPT-I may be mediated (at least in part) by increased phosphorylation and subsequent activation of Ca2+/CM-PKII. PMID:9003421

  5. The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization.

    Science.gov (United States)

    Licata, Stephanie C; Pierce, R Christopher

    2003-04-01

    Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. PMID:12641723

  6. Human Calmodulin-Like Protein CALML3: A Novel Marker for Normal Oral Squamous Mucosa That Is Downregulated in Malignant Transformation

    Directory of Open Access Journals (Sweden)

    Michael D. Brooks

    2013-01-01

    Full Text Available Oral cancer is often diagnosed only at advanced stages due to a lack of reliable disease markers. The purpose of this study was to determine if the epithelial-specific human calmodulin-like protein (CALML3 could be used as marker for the various phases of oral tumor progression. Immunohistochemical analysis using an affinity-purified CALML3 antibody was performed on biopsy-confirmed oral tissue samples representing these phases. A total of 90 tissue specimens were derived from 52 patients. Each specimen was analyzed in the superficial and basal mucosal cell layers for overall staining and staining of cellular subcompartments. CALML3 was strongly expressed in benign oral mucosal cells with downregulation of expression as squamous cells progress to invasive carcinoma. Based on the Cochran-Armitage test for trend, expression in the nucleus and at the cytoplasmic membrane significantly decreased with increasing disease severity. Chi-square test showed that benign tissue specimens had significantly more expression compared to dysplasia/CIS and invasive specimens. Dysplasia/CIS tissue had significantly more expression than invasive tissue. We conclude that CALML3 is expressed in benign oral mucosal cells with a statistically significant trend in downregulation as tumorigenesis occurs. CALML3 may thus be a sensitive new marker for oral cancer screening.

  7. Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

    Science.gov (United States)

    Piotrowski, Christine; Ihling, Christian H; Sinz, Andrea

    2015-11-01

    Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner. PMID:25726908

  8. Variants in doublecortin- and calmodulin kinase like 1, a gene up-regulated by BDNF, are associated with memory and general cognitive abilities.

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    Stéphanie Le Hellard

    Full Text Available BACKGROUND: Human memory and general cognitive abilities are complex functions of high heritability and wide variability in the population. The brain-derived neurotrophic factor (BDNF plays an important role in mammalian memory formation. METHODOLOGY / PRINCIPAL FINDING: Based on the identification of genes markedly up-regulated during BDNF-induced synaptic consolidation in the hippocampus, we selected genetic variants that were tested in three independent samples, from Norway and Scotland, of adult individuals examined for cognitive abilities. In all samples, we show that markers in the doublecortin- and calmodulin kinase like 1 (DCLK1 gene, are significantly associated with general cognition (IQ scores and verbal memory function, resisting multiple testing. DCLK1 is a complex gene with multiple transcripts which vary in expression and function. We show that the short variants are all up-regulated after BDNF treatment in the rat hippocampus, and that they are expressed in the adult human brain (mostly in cortices and hippocampus. We demonstrate that several of the associated variants are located in potential alternative promoter- and cis-regulatory elements of the gene and that they affect BDNF-mediated expression of short DCLK1 transcripts in a reporter system. CONCLUSION: These data present DCLK1 as a functionally pertinent gene involved in human memory and cognitive functions.

  9. Chronic hyperammonemia reduces the activity of neuronal nitric oxide synthase in cerebellum by altering its localization and increasing its phosphorylation by calcium-calmodulin kinase II.

    Science.gov (United States)

    El-Mlili, Nisrin; Rodrigo, Regina; Naghizadeh, Bahareh; Cauli, Omar; Felipo, Vicente

    2008-08-01

    Impaired function of the glutamate-nitric oxide-cGMP pathway contributes to cognitive impairment in hyperammonemia and hepatic encephalopathy. The mechanisms by which hyperammonemia impairs this pathway remain unclear. Understanding these mechanisms would allow designing clinical treatments for cognitive deficits in hepatic encephalopathy. The aims of this work were: (i) to assess whether chronic hyperammonemia in vivo alters basal activity of neuronal nitric oxide synthase (nNOS) in cerebellum and/or its activation in response to NMDA receptor activation and (ii) to analyse the molecular mechanisms by which hyperammonemia induces these alterations. It is shown that hyperammonemia reduces both basal activity of nNOS and its activation following NMDA receptor activation. Reduced basal activity is because of increased phosphorylation in Ser847 (by 69%) which reduces basal activity of nNOS by about 40%. Increased phosphorylation of nNOS in Ser847 is because of increased activity of calcium-calmodulin-dependent protein kinases (CaMKII) which in turn is because of increased phosphorylation at Thr286. Inhibiting CaMKII with KN-62 normalizes phosphorylation of Ser847 and basal NOS activity in hyperammonemic rats, returning to values similar to controls. Reduced activation of nNOS in response to NMDA receptor activation in hyperammonemia is because of altered subcellular localization of nNOS, with reduced amount in post-synaptic membranes and increased amount in the cytosol. PMID:18498443

  10. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

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    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  11. Calcium/calmodulin-dependent kinase IV contributes to translation-dependent early synaptic potentiation in the anterior cingulate cortex of adult mice

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    Toyoda Hiroki

    2010-09-01

    Full Text Available Abstract Calcium/calmodulin-dependent kinase IV (CaMKIV phosphorylates the major transcription factor, cyclic AMP-responsive element binding protein (CREB, which plays key roles in synaptic plasticity and memory consolidation. Our previous study showed that long-term potentiation (LTP in the anterior cingulate cortex (ACC was significantly enhanced in transgenic mice overexpressing CaMKIV. Considering that the CaMKIV-CREB pathway plays a central role in the protein synthesis-dependent LTP, it is possible that upregulation of CaMKIV contributes to enhancement of LTP by promoting protein synthesis. To test this possibility, we examined the effects of transcription and translation inhibitors on synaptic potentiation induced by pairing of synaptic activity with postsynaptic depolarization (paired training in ACC pyramidal neurons of wild-type and CaMKIV transgenic mice. We found that synaptic potentiation induced by paired training was partially inhibited by transcription or translation inhibitors both in wild-type and CaMKIV transgenic mice; the extent of inhibition was markedly larger in the CaMKIV transgenic mice than in the wild-type mice. Biochemical and immunohistochemical studies revealed that CaMKIV was distributed in the membrane, cytosol and nucleus of ACC neurons. Our results reveal in the first time a transcription- and translation-dependent component of early synaptic LTP in adult ACC synapses, and demonstrate that CaMKIV enhances early synaptic potentiation by activating new protein synthesis.

  12. Apo-states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain

    Science.gov (United States)

    Findeisen, Felix; Rumpf, Christine; Minor, Daniel L.

    2013-01-01

    In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation (CDI) and limits calcium entry, whereas CaBP1 blocks CDI and allows sustained calcium influx. Here, we combine isothermal titration calorimetry (ITC) with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium binding properties. The observation that the apo-forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. PMID:23811053

  13. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    International Nuclear Information System (INIS)

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate

  14. Ca2+/calmodulin-dependent protein kinase kinase is not involved in hypothalamic AMP-activated protein kinase activation by neuroglucopenia.

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    Junji Kawashima

    Full Text Available Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca(2+/Calmodulin-dependent protein kinase kinase (CaMKK, which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO, a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV in rats 30 min before 2DG ICV injection (40 µmol to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK.

  15. Functional properties of the CaV1.2 calcium channel activated by calmodulin in the absence of alpha2delta subunits.

    Science.gov (United States)

    Ravindran, Arippa; Kobrinsky, Evgeny; Lao, Qi Zong; Soldatov, Nikolai M

    2009-01-01

    Voltage-activated CaV1.2 calcium channels require association of the pore-forming alpha1C subunit with accessory CaVbeta and alpha2delta subunits. Binding of a single calmodulin (CaM) to alpha1C supports Ca2+-dependent inactivation (CDI). The human CaV1.2 channel is silent in the absence of CaVbeta and/or alpha2delta. Recently, we found that coexpression of exogenous CaM (CaMex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of CaVbeta. Here we discovered that CaMex and its Ca2+-insensitive mutant (CaM1234) rendered active alpha1C/CaVbeta channel in the absence of alpha2delta. Coexpression of CaMex with alpha1C and beta2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by approximately +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca2+) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaMex and CaM1234 accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal CaV1.2 channel. The data suggest a previously unknown action of CaM that in the presence of CaVbeta; translates into activation of the alpha2delta-deficient calcium channel and alteration of its properties. PMID:19106618

  16. Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

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    Rajesh Bhardwaj

    2013-10-01

    Full Text Available Ca2+ (calcium homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum and Ca2+ channels in the PM (plasma membrane. STIM1 (stromal interaction molecule 1 and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.

  17. Chronic ethanol intake-induced changes in open-field behavior and calcium/calmodulin-dependent protein kinase Ⅳ expression in nucleus accumbens of rats: naloxone reversal

    Institute of Scientific and Technical Information of China (English)

    Jing LI; Wei-liang BIAN; Gui-qin XIE; Sheng-zhong CUI; Mei-ling WU; Yue-hua LI; Ling-li QUE; Xiao-ru YUAN

    2008-01-01

    Aim: To investigate the effects of chronic ethanol intake on the locomotor activity and the levels of calcium/calmodulin-dependent protein kinase Ⅳ (CaM kinase Ⅳ) in the nucleus accumbens (NAc) of rats. Simultaneously, the effects of non-selective opioid antagonist (naloxone) on the CaM kinase Ⅳ expression in the NAc and ethanol consumption of rats were also observed. Methods: Ethanol was administered in drinking water at the concentrations of 6% (v/v), for 28 d. The locomotor activity of rats was investigated in the open-field apparatus. CaM kinase Ⅳ levels in the NAc were analyzed using Western blotting. Results: Rats consuming ethanol solution exhibited a significant decrease of ambulation activity, accompanied by a reduced frequency of explorative rearing in an open-field task on d 7 and d 14 of chronic ethanol ingestion, whereas presumed adaptation to the neurological effects of ethanol was observed on d 28. Chronic ethanol intake elicited a significant decrease of the CaM kinase Ⅳ expression in the nuclei, but not in the cytoplasm of the NAc on d 28. Naloxone treatment significantly attenu-ated ethanol intake of rats and antagonized the decrease of CaM kinase Ⅳ in the nuclei of NAc neurons. The cytosolic CaM kinase Ⅳ protein levels of the NAc also increased in rats exposed to ethanol plus naloxone. Conclusion: Chronic ethanol intake-induced changes in explorative behavior is mediated at least partly by changes in CaM kinase Ⅳ signaling in the nuclei of the NAc, and naloxone attenuates ethanol consumption through antagonizing the downregulation of CaM kinase Ⅳ in the NAc.

  18. Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca 2+ -calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jiusheng; van den Bedem, Henry; Brunger, Axel T.; Wilson, Mark A.

    2016-01-01

    Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

  19. The roles and relations of calpastatin, calmodulin and an undefined cytoplasmic factor in the regulation of cardiac L-type Ca2+ channels

    Institute of Scientific and Technical Information of China (English)

    HAO Li-ying; ZHU Tong; HU Hui-yuan; ZHAO Mei-mi; RUI Feng; LIU Yan; ZHAO Jin-sheng; tsuko Minobe; Masaki Kameyama

    2008-01-01

    Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down'. The factors include ATP, calpastatin and H fraction (a high molecular fraction of bovine cardiac cytoplasm). Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes. Run-down was induced by the inside-out patch formation. Calpastatin (CS), calmodulin(CaM) and three GST-fusion fragment peptides derived from the C-terminal tail of guineapig Car1.2, CT-1 (amino acids number 1509-1791), CTo2 (1777-2003) and CT-3 (1944-2169) were produced as GST fusion proteins. Results (1)CaM + ATP or CS + ATP restored the channels after rundown;however, the CaM or CS's effects became smaller with the longer run-down time. (2)After run down, CaM-dependent protein kinase (CaMKII) produced Ca2+ channel activity to only 2-10% of the basal activity, however, in the presence of CaMKII, the time-dependent nature of the CaM effect was abolished. (3) In pull-down assay, CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase. (4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm. Conclusions The results show that CS, CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels, and that there might be cross talks among the four factors (CS, CaM, CaMKII and the undefined cytoplasmic factor). This work was supported by the grants from the Japan Society for the Promotion of Science and the National Natural Science Foundation of China (No. 30670761, No. 30671726).

  20. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2005-06-24

    Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium. PMID:15896312

  1. Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

    Science.gov (United States)

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

    2014-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. PMID:25064143

  2. Regulation of Voltage-Gated Ca2+ Currents by Ca2+/Calmodulin-dependent Protein Kinase II in Resting Sensory Neurons

    Science.gov (United States)

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H.

    2014-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca2+ channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca2+ currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16 – 30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. PMID:25064143

  3. Molecular cloning and bioinformatics analysis of calmodulin genes in Phenacoccus solenopsis Tinsley%扶桑绵粉蚧钙调蛋白基因的克隆与生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    罗梅; 董章勇; 宾淑英; 林进添; 吴仲真; 李献锋

    2012-01-01

    Calmodulin(CaM) plays an important regulatory role in a variety of in vivo Ca2 +-dependent cell functions and enzyme systems. It was the first time to clone the calmodulin gene from Phe-nacoccus solenopsis Tinsley. The full-length of open reading frame (ORF) is 447 bp, encoding 148 ami-no acid residues. PsCaM gene was constituted by three introns and four exons. The intron lengths were 73,81,72 bp; The lengths of the 4 separated exons were 33,133,183,98 bp. Functional domain analysis showed that the protein got two EF-hand domains, with 13 Ca2+ binding sites. The theoretical protein isoelectric point is 6. 21. It was stable protein, with no transmembrane region. The three-dimensional structure of its protein was obtained by homology modeling. Multiple sequence comparison revealed that the gene is relatively conservative. The research was the basis for further study of the functional mechanisms of calmodulin genes.%钙调蛋白(calmodulin,CaM)对生物体内多种Ca2+依赖的细胞功能和酶体系都有重要的调节作用.为研究扶桑绵粉蚧的信号转导受体蛋白,首次克隆了扶桑绵粉蚧钙调蛋白基因PsCaM的cDNA全长序列,其开放阅读框(ORF)包含447 bp的片段,编码148个氨基酸.PsCaM基因由3个内含子和4个外显子组成.3个内含子的长度分别为73、81、72 bp,分隔的4个外显子的长度分别为33、133、183、98 bp.功能域分析结果显示:该蛋白具有2个EF-hand结构域,有13个Ca2+结合位点;该蛋白的理论等电点是6.21,属于稳定蛋白,且没有跨膜区域;通过同源建模获得了其蛋白的三维结构.多序列比较显示,P sCaM基因相对较保守.

  4. Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C.

    Science.gov (United States)

    Raufman, J P; Malhotra, R; Xie, Q; Raffaniello, R D

    1997-03-01

    In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells

  5. The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

    OpenAIRE

    Taiakina, Valentina; Boone, Adrienne N.; Fux, Julia; Senatore, Adriano; Weber-Adrian, Danielle; Guillemette, J. Guy; Spafford, J. David

    2013-01-01

    NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing...

  6. Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges

    OpenAIRE

    Blair, Robert E.; Sombati, Sompong; Churn, Severn B.; DeLorenzo, Robert J.

    2008-01-01

    Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM Kinase II have not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long lasting decrease in CaM Kinase II activity in the hippocampal neuronal culture model of low Mg2+ induced spontaneous recurrent...

  7. Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.

    Science.gov (United States)

    Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana

    2016-05-13

    Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network. PMID:26987903

  8. Distinct properties of Ca2+-calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Sze-Yi; Procko, Erik; Gaudet, Rachelle [Harvard

    2012-11-01

    Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca2+–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca2+–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca2+–CaM, respectively. This structure is similar to canonical Ca2+–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca2+–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca2+–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca2+–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.

  9. Extending students' practice of metacognitive regulation strategies in the undergraduate chemistry laboratory and investigation of Pb2+ binding to calmodulin with circular dichroism and molecular dynamics modeling

    Science.gov (United States)

    Valencia Navarro, Laura N.

    The following dissertation was composed of two projects in chemistry education and benchwork/computational biochemistry. The chemistry education research explored students' practice of metacognitive strategies while solving open-ended laboratory problems when engaged in an instructional environment, the Science Writing Heuristic (SWH), that was characterized as supporting metacognitive regulation strategy use. Through in-depth interviews with students, results demonstrated that students in the SWH environment, compared to non-SWH students, used metacognitive strategies to a greater degree and to a greater depth when solving open-ended laboratory problems. As students engaged in higher levels of metacognitive regulation, their elective use of peers became a prominent path for supporting the practice of metacognitive strategies. Students claimed that the structure of the SWH weekly laboratory experiments improved their ability to solve open-ended lab problems. This research not only provided a lens into students' descriptions of their regulation strategy practices in the laboratory, but it also supported that the way that a laboratory environment is arranged can affect these regulation strategy practices and their transfer to new situations. In the biochemical study on the binding of Pb2+ to calmodulin (CaM), data was acquired via circular dichroism (CD) and molecular dynamics modeling. CD signal data indicated a unique signal from Pb-CaM and a significantly smaller ratio theta208/theta222 for Pb-CaM than Ca-CaM. An analysis of secondary structure content indicated that alpha-helical structure decreased and random coil structure increased when CaM was saturated with Pb2+ compared to Ca2+ saturated CaM. A molecular dynamics simulation of Pb2+ binding to CaM showed that Pb2+ ions bound to sites outside of the known canonical binding sites including the linker region, and indicated change in secondary structure. These results support the theory of opportunistic binding

  10. Effect of Calcium Ion Removal, Ionic Strength, and Temperature on the Conformation Change in Calmodulin Protein at Physiological pH

    Directory of Open Access Journals (Sweden)

    Sunita Negi

    2014-01-01

    Full Text Available The response of the calmodulin (CaM protein as a function of calcium ion removal, ionic strength, and temperature at physiological pH condition was investigated using classical molecular dynamics simulations. Changing the ionic strength and temperature came out to be two of the possible routes for observing a conformation change in the protein. This behavior is similar to the conformation change observed in our previous study where a change in the pH was observed to trigger a conformation change in this protein. In the present study, as the calcium ions are removed from the protein, the protein is observed to acquire more flexibility. This flexibility is observed to be more prominent at a higher ionic strength. At a lower ionic strength of 150 mM with all the four calcium ions intact, the N- and C-lobes are observed to come close to a distance of 30 Å starting from an initial separation distance of 48 Å. This conformation change is observed to take place around 50 ns in a simulation of 100 ns. As a second parameter, temperature is observed to play a key role in the conformation change of the protein. With an increase in the temperature, the protein is observed to acquire a more compact form with the formation of different salt bridges between the residues of the N- and the C-lobes. The salt bridge formation leads to an overall lowering of the energy of the protein thus favoring the bending of the two lobes towards each other. The improper and dihedral terms show a significant shift thus leading to a more compact form on increasing the temperature. Another set of simulations is also performed at an increased temperature of 500 K to verify the reproducibility of the results. Thus a set of three possible alterations in the environmental conditions of the protein CaM are studied, with two of them giving rise to a conformation change and one adding flexibility to the protein.

  11. Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.

    Directory of Open Access Journals (Sweden)

    Dan Zhang

    Full Text Available BACKGROUND: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. METHODOLOGY/PRINCIPAL FINDINGS: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR or Catestatin (CAT induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate, a store operated channels (SOCs blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. CONCLUSIONS/SIGNIFICANCE: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated i

  12. Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

    Directory of Open Access Journals (Sweden)

    Hsieh Hsi-Lung

    2010-11-01

    Full Text Available Abstract Background Lipoteichoic acid (LTA is a component of Gram-positive bacterial cell walls, which has been found to be elevated in cerebrospinal fluid of patients suffering from meningitis. Moreover, matrix metalloproteinases (MMPs, MMP-9 especially, have been observed in patients with brain inflammatory diseases and may contribute to brain disease pathology. However, the molecular mechanisms underlying LTA-induced MMP-9 expression in brain astrocytes remain unclear. Objective The goal of this study was to examine whether LTA-induced cell migration is mediated by calcium/calmodulin (CaM/CaM kinase II (CaMKII-dependent transactivation of the PDGFR pathway in rat brain astrocytes (RBA-1 cells. Methods Expression and activity of MMP-9 induced by LTA was evaluated by zymographic, western blotting, and RT-PCR analyses. MMP-9 regulatory signaling pathways were investigated by treatment with pharmacological inhibitors or using dominant negative mutants or short hairpin RNA (shRNA transfection, and chromatin immunoprecipitation (ChIP-PCR and promoter activity reporter assays. Finally, we determined the cell functional changes by cell migration assay. Results The data show that c-Jun/AP-1 mediates LTA-induced MMP-9 expression in RBA-1 cells. Next, we demonstrated that LTA induces MMP-9 expression via a calcium/CaM/CaMKII-dependent transactivation of PDGFR pathway. Transactivation of PDGFR led to activation of PI3K/Akt and JNK1/2 and then activated c-Jun/AP-1 signaling. Activated-c-Jun bound to the AP-1-binding site of the MMP-9 promoter, and thereby turned on transcription of MMP-9. Eventually, up-regulation of MMP-9 by LTA enhanced cell migration of astrocytes. Conclusions These results demonstrate that in RBA-1 cells, activation of c-Jun/AP-1 by a CaMKII-dependent PI3K/Akt-JNK activation mediated through transactivation of PDGFR is essential for up-regulation of MMP-9 and cell migration induced by LTA. Understanding the regulatory mechanisms

  13. Clozapine functions through the prefrontal cortex serotonin 1A receptor to heighten neuronal activity via calmodulin kinase II-NMDA receptor interactions.

    Science.gov (United States)

    Purkayastha, Sudarshana; Ford, Jason; Kanjilal, Baishali; Diallo, Souleymane; Del Rosario Inigo, Joseph; Neuwirth, Lorenz; El Idrissi, Abdeslem; Ahmed, Zaghloul; Wieraszko, Andrzej; Azmitia, Efrain C; Banerjee, Probal

    2012-02-01

    Aberrant dopamine release in the prefrontal cortex (PFC) is believed to underlie schizophrenia, but the mechanistic pathway through which a widely used antipsychotic, clozapine (Clz), evokes neurotransmitter-releasing electrical stimulation is unclear. We analyzed Clz-evoked regulation of neuronal activity in the PFC by stimulating axons in layers IV and V and recording the electrical effect in the post-synaptic pyramidal cells of layers II and III. We observed a Clz-evoked increase in population spike (PS), which was mediated by serotonin 1A receptor (5-HT(1A)-R), phospholipase Cβ, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Immunoblotting demonstrated that the Clz-activation of CaMKII was 5-HT(1A)-R-mediated. Intriguingly, the NMDA receptor (NMDA-R) antagonist (±)2-amino-5-phosphonovaleric acid (APV) eliminated the Clz-mediated increase in PS, suggesting that the 5-HT(1A)-R, NMDA-R and CaMKII form a synergistic triad, which boosts excitatory post-synaptic potential (EPSP), thereby enhancing PS. In corroboration, Clz as well as NMDA augmented field EPSP (fEPSP), and WAY100635 (a 5-HT(1A)-R antagonist), APV, and a CaMKII inhibitor eliminated this increase. As previously shown, CaMKII binds to the NMDA-R 2B (NR2B) subunit to become constitutively active, thereby inducing α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor recruitment to the post-synaptic membrane and an increase in fEPSP. Co-immunoprecipitation demonstrated that Clz potentiates interactions among CaMKII, NR2B, and 5-HT(1A)-R, possibly in the membrane rafts of the post-synaptic density (PSD), because pretreatment with methyl-β-cyclodextrin (MCD), an agent that disrupts rafts, inhibited both co-immunoprecipitation as well as fEPSP. In summary, Clz functions in the PFC by orchestrating a synergism among 5-HT(1A)-R, CaMKII, and NMDA-R, which augments excitability in the PFC neurons of layers II/III. PMID:22044428

  14. Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges.

    Science.gov (United States)

    Blair, Robert E; Sombati, Sompong; Churn, Severn B; Delorenzo, Robert J

    2008-06-24

    Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model. PMID:18495112

  15. Immunoselection of cDNAs to avian intestinal calcium binding protein 28K and a novel calmodulin-like protein: assessment of mRNA regulation by the Vitamin D hormone

    International Nuclear Information System (INIS)

    Calcium's role in a variety of cellular processes has been well documented. The storage, distribution, and delivery of calcium are regulated by a family of binding proteins including troponin C, calmodulin, parvalbumin, and vitamin D dependent calcium binding protein (CaBP-28), all of which have evolved from a common ancestral gene. To evaluate vitamin D regulation of gene transcription, a CaBP-28 cDNA (767 base pairs) was isolated from a chicken intestine λgt11 library utilizing a polyvalent CaBP-28 antibody as a probe. Coincident with the identification of the CaBP-28 cDNA, a group of cDNAs also was isolated (with the anti-CaBP-28 antibody) that demonstrated 84% nucleotide homology and 99% deduced amino acid homology with chicken brain calmodulin (CaM). This new CaM-like cDNA was named neoCaM. There is little nucleotide homology between the CaBP-28 cDNA and neoCaM. The CaBP-28 cDNA hybridizes with three transcripts of 2000, 2900, and 3300 bases which are dramatically induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], while the neoCaM cDNA recognizes three distinct (from CaBP-28) transcripts. Two of these mRNAs are 1400 and 1800 bases as described for brain CaM, but another large 4000-base transcript is detected with neoCaM. Neither the CaM nor the neoCaM transcript reveals any modulation by 1,25(OH)2D3. Herein, the authors discuss the possible significance of not only the isolation of both cDNAs with a single antibody but also the relation of neoCaM to other well-characterized CaM cDNAs

  16. The Mechanism of Calcium and Calcium-activated Calmodulin Modulating the Expression of Hsp70%钙-钙调素信号通路调节热应激蛋白70表达的机制

    Institute of Scientific and Technical Information of China (English)

    齐晓楠; 赵园; 田文儒

    2015-01-01

    随着温室效应的加剧,全球气候变暖已经成为现代畜牧业生产体系所面临的严峻挑战.高温炎热气候已成为影响畜禽生长繁殖的一种主要的非生物胁迫.热应激刺激时,动物体产生一系列生理变化来提高机体的耐受性,而本文旨在阐述钙(calcium,Ca2+)-钙调蛋白(calcium-activated calmodulin,CaM)参与的耐热信号的转导机制,即热应激蛋白(heat shock proteins,Hsps)的表达机制.热应激时,细胞内Ca2+浓度上升,激活CaM及其表达量的增加,随即CaM作用于其下游的2种靶蛋白,即2条信号转导支路来提高热激转录因子l(heat shock factor 1,HSFl)的转录活性,进而调节热应激蛋白70(Hsp70)的表达.热应激时,Hsps表达的2条具体通路:①Ca2+-CaM激活下游的靶蛋白—钙/钙调素依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ),CaMKⅡ通过促进HSF1(230)的磷酸化来提高HSF1的转录活性,进而诱导Hsp70的表达;②Ca2+-CaM激活下游另一个靶蛋白Hsp70,Hsp70通过与增多的CaM形成复合物,释放HSF1单体,增多的HSF1形成有活性的HSF1三聚体,并入核与热激元件(heat shockelement,HSE)结合,使HSF1具有转录活性,进而诱导Hsp70的表达.本文系统完整地综述了Ca2+-CaM-HSF1-Hsp70热应激信号系统,在感受、传导和响应热应激刺激时的机制.

  17. The calmodulin-like proteins AtCML4 and AtCML5 are single-pass membrane proteins targeted to the endomembrane system by an N-terminal signal anchor sequence.

    Science.gov (United States)

    Ruge, Henning; Flosdorff, Sandra; Ebersberger, Ingo; Chigri, Fatima; Vothknecht, Ute C

    2016-06-01

    Calmodulins (CaMs) are important mediators of Ca(2+) signals that are found ubiquitously in all eukaryotic organisms. Plants contain a unique family of calmodulin-like proteins (CMLs) that exhibit greater sequence variance compared to canonical CaMs. The Arabidopsis thaliana proteins AtCML4 and AtCML5 are members of CML subfamily VII and possess a CaM domain comprising the characteristic double pair of EF-hands, but they are distinguished from other members of this subfamily and from canonical CaMs by an N-terminal extension of their amino acid sequence. Transient expression of yellow fluorescent protein-tagged AtCML4 and AtCML5 under a 35S-promoter in Nicotiana benthamiana leaf cells revealed a spherical fluorescence pattern. This pattern was confirmed by transient expression in Arabidopsis protoplasts under the native promoter. Co-localization analyses with various endomembrane marker proteins suggest that AtCML4 and AtCML5 are localized to vesicular structures in the interphase between Golgi and the endosomal system. Further studies revealed AtCML5 to be a single-pass membrane protein that is targeted into the endomembrane system by an N-terminal signal anchor sequence. Self-assembly green fluorescent protein and protease protection assays support a topology with the CaM domain exposed to the cytosolic surface and not the lumen of the vesicles, indicating that AtCML5 could sense Ca(2+) signals in the cytosol. Phylogenetic analysis suggests that AtCML4 and AtCML5 are closely related paralogues originating from a duplication event within the Brassicaceae family. CML4/5-like proteins seem to be universally present in eudicots but are absent in some monocots. Together these results show that CML4/5-like proteins represent a flowering plant-specific subfamily of CMLs with a potential function in vesicle transport within the plant endomembrane system. PMID:27029353

  18. The Golgi α-1,6 mannosyltransferase KlOch1p of Kluyveromyces lactis is required for Ca2+/calmodulin-based signaling and for proper mitochondrial functionality

    Directory of Open Access Journals (Sweden)

    Mancini Patrizia

    2009-12-01

    Full Text Available Abstract Background Protein N-glycosylation is a relevant metabolic pathway in eukaryotes and plays key roles in cell processes. In yeasts, outer chain branching is initiated in the Golgi apparatus by the alpha-1,6-mannosyltransferase Och1p. Results Here we report that, in Kluyveromyces lactis, this glycosyltransferase is also required to maintain functional mitochondria and calcium homeostasis. Cells carrying a mutation in KlOCH1 gene showed altered mitochondrial morphology, increased accumulation of ROS and reduced expression of calcium signalling genes such as calmodulin and calcineurin. Intracellular calcium concentration was also reduced in the mutant cells with respect to the wild type counterparts. Phenotypes that occur in cells lacking the alpha-1,6-mannosyltransferase, including oxidative stress and impaired mitochondria functionality, were suppressed by increased dosage of KlCmd1p. This, in turn, acts through the action of calcineurin. Conclusions Proper functioning of the alpha-1,6-mannosyltransferase in the N-glycosylation pathway of K. lactis is required for maintaining normal calcium homeostasis; this is necessary for physiological mitochondria dynamics and functionality.

  19. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

    Directory of Open Access Journals (Sweden)

    Valentina Taiakina

    Full Text Available NSCaTE is a short linear motif of (xWxxx(I or Lxxxx, composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA, but disappears in high buffer conditions (10 mM EGTA. Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

  20. Calmodulin antagonists have differential effects on Ca/sup 2 +/ uptake, (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase and Ca/sup 2 +/ release in hepatic endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Delfert, D.M.; Koepnick, S.; McDonald, J.M.; Chan, K.M.

    1986-05-01

    The effect of calmodulin (CaM) antagonists on Ca/sup 2 +/ handling by hepatic endoplasmic reticulum (ER) was studied. Ca/sup 2 +/ uptake by saponin-permeabilized hepatocytes or isolated ER was measured using /sup 45/Ca/sup 2 +/ in a filtration assay in the presence of 0.09 ..mu..M free (Ca/sup 2 +/) and inhibitors of mitochondrial Ca/sup 2 +/ transport. Each CaM-antagonist (chlorpromazine, CPZ; trifluoperazine, TFP; calmidazolium, W7 and 48/80) showed a dose-dependent inhibition of Ca/sup 2 +/ accumulation in permeabilized hepatocytes. Both the initial rate and steady state values for Ca/sup 2 +/ uptake were reduced by 50% with 40 ..mu..M calmidazolium, 100 ..mu..M TFP, 150..mu..M W7, 150 ..mu..M CPZ and 300 ..mu..M 48/80. Using isolated ER both calmidazolium (20 ..mu..M) and W7 (150 ..mu..M) inhibited the initial rate and steady state level of Ca/sup 2 +/ accumulation. At this concentration calmidazolium inhibited the initial rate of (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase activity, and enhanced Ca/sup 2 +/ release. In contrast, W7 had no effect on these parameters. These results suggest that the reduced level of Ca/sup 2 +/ uptake into ER vesicles in the presence of calmidazolium may result from inhibition of the (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase as well as induction of Ca/sup 2 +/ release, while W7 may act to uncouple Ca/sup 2 +/ transport from its (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase counterpart.

  1. Roles of Calmodulin-dependent Protein Kinase Ⅱ in Meiotic Maturation and Fertilization of Oocytes%钙调蛋白依赖的蛋白激酶Ⅱ在卵母细胞减数分裂和受精中的作用

    Institute of Scientific and Technical Information of China (English)

    范衡宇; 霍立军; 孙青原

    2003-01-01

    钙调蛋白依赖的蛋白激酶(CaMK)是一类分布广泛的丝/苏氨酸蛋白激酶家族,在钙离子和钙调蛋白存在的条件下发生自磷酸化而被激活,在细胞内对于钙信号的传递具有重要的介导作用.近年来的研究表明CaMKⅡ是参与调节卵母细胞减数分裂的重要分子,在卵母细胞成熟、极体排放、受精和活化等过程中发挥作用.CaMKⅡ作为Ca2+的下游信号分子,在受精后促进成熟促进因子(MPF)和细胞静止因子(CSF)的失活,并调节纺锤体微管的组装和中心体的复制过程.虽然CaMKⅡ在减数分裂中的作用广泛而关键,但目前的研究主要集中于低等动物和小鼠,今后有待进一步阐明该蛋白激酶在其他哺乳动物中的作用和调节机制.%Calmodulin-dependent protein kinase (CaMK), activated by auto-phosphorylation at the presence of calcium and calmodulin, is widely distributed in eukaryotes. CaMKs are important mediators of calcium signal in eukaryotes. Recent researches have suggested that CaMKⅡ is involved in the regulation of meiotic cell cycle of oocytes. It plays functional roles in meiotic maturation, polar body extrusion, fertilization and egg activation. As one of the down-stream signaling molecules of calcium, CaMKⅡ facilitates the inactivation of maturation promoting factor (MPF) and cytostatic factor (CSF) following fertilization, as well as the spindle microtubule organization and centrosome duplication. Although the functions of CaMKⅡ in oocyte meiosis are versatile and essential, the present results are primarily obtained from low vertebrates and mouse. In future studies, the function and regulation of this kinase in other mammals should be stressed.

  2. 钙/钙调素依赖性蛋白激酶Ⅱ对热休克小鼠胚胎成纤维细胞HSP70表达的影响%Calcium/Calmodulin-dependent Protein Kinase Ⅱ Contributes to HSP70 Expression in Mouse Embryonic Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    孙琪; 丛霞; 索佳佳; 曹荣峰; 姜忠玲; 崔凯; 高善颂; 田文儒

    2012-01-01

    The objectives of the study were to verify if the CaMK II (Ca +/calmodulin-dependent protein kinase II ) influences the expression of HSP70 gene in the mouse embryonic fibroblasts (MEF) with heat shock and to clarify its mechanism. The fibroblasts were heat-treated at 37t ,39^ and 41°C individually before the expressions of both CaMK II and HSF1 were detected by RT-PCR at 0. 5 ,1 ,1. 5 and 2 h of heat shock ,respectivelly. Moreover, the fibroblasts were divided into control group and myr-AIP group randomly,and cultured at 371 and 39°C respectively. The expressions of both HSP70 and HSF1 were detected. The expressions of HSF1 and CaMK II mRNA in the MEF treated at 39^ for 1 h were both extremely significantly higher (P <0. 01) than that of the control group. There was a significantly (P <0. 05) decrease of HSP70 expression in the MEF cultured at 39t treated with myr-AIP compared with its blank control. However, there was no difference in HSP70 exoression between the MEF cultured at 37X1 with or without myr-AIP. The expressions of HSF1 in the MEF treated with myr-AIP were not significantly influenced. However,the p-HSFl expression in the MEF treated with myr-AIP was significantly (P <0. 05) decreased. Moderate heat shock increases both expressions of CaMK FJ and HSF1 in mouse embryonic fibroblasts. CaMK II participates in both heat shock response and HSP70 gene expression by p-HSFl.%为证实热休克小鼠胎儿成纤维细胞中钙/钙调素依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)对热休克蛋白70 (Heat shock protein70,HSP70)基因表达的影响及其作用机理,将体外培养的小鼠胎儿成纤维细胞(Mouse Embryonic Fibroblasts,MEF)随机分为39℃和41℃热处理组(分别处理0.5,1,1.5,2h)和常温对照组(37℃),测定各组细胞CaMKⅡ和HSF1 mRNA的量.此外,将培养的细胞分为37℃和39℃CaMKⅡ特异性抑制剂(myr-AIP)处理组和相应的空白对照组,分别测定各组细胞HSP70、HSF1

  3. Effect of calmodulin inhibitor on NO production and iNOS expression induced by LPS/IFN-γin RAW264.7 cells%CaM抑制剂对LPS/IFN-γ诱导的RAW264.7细胞NO生成及iNOS表达的影响

    Institute of Scientific and Technical Information of China (English)

    袁小媚; 雷寒; 柳青; 夏勇; 马康华

    2011-01-01

    Objective To investigate the effect of calmodulin (CaM) inhibitor on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression induced by lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) in mouse RAW264.7 macrophage cells, and to study the anti-inflammation mechanism of CaM inhibitor. Methods An inflammatory response model was established using RAW264.7 cells induced by LPS and/or IFN-γ. CaM inhibitors N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) and triflu operazine (TFP) were used to pre-treat RAW264.7 cells to evaluate the effects of the CaM inhibitors. NO con centration was measured with Griess Reagent Kit. Target protein and mRNA expression was tested by Western blot and RT-PCR. Results In RAW264.7 cells induced by LPS or IFN-γ CaM inhibitors W-7 and TFP sig nificantly blocked NO production ( P < 0. 01 ) and protein and mRNA expression of iNOS ( P < 0. 01 ). In RAW264.7 cells induced by LPS and IFN-γ, W-7 and TFP remarkably prevented IKBα degradation (P < 0. 01 ) and phospho-IKK and phospho-STAT1 protein expression ( P < 0. 01 ). Conclusion CaM inhibitors W-7 and TFP can inhibit LPS/IFN-γ-induced NO production and iNOS protein and mRNA expression in RAW264.7 cells, probably by the mechanism of suppressing IKBα degradation and phospho-IKK and phospho- STAT1 protein expression. The anti-inflammatory effects of CaM inhibitors may be associated with NF-KB and JAK-STAT1 signaling pathways.%目的 观察钙调蛋白(Calmodulin,CaM)抑制剂对脂多糖/干扰素γ(LPS/IFN-γ)诱导的RAW264.7细胞一氧化氮(NO)的生成及诱生型一氧化氮合酶(iNOS)表达的作用及其可能的作用机制.方法 采用LPS/IFN-γ诱导RAW264.7巨噬细胞建立细胞炎症反应模型,钙调蛋白抑制剂(W-7,TFP)预处理细胞后,采用Griess试剂法测定NO释放量;采用Western blot法测定目的 蛋白的表达;采用反转录聚合酶链反应法(RT-PCR)分析iNOS mRNA表达的变化.结果 CaM抑制剂

  4. Inhibition of endogenous heat shock protein 70 attenuates inducible nitric oxide synthase induction via disruption of heat shock protein 70/Na(+) /H(+) exchanger 1-Ca(2+) -calcium-calmodulin-dependent protein kinase II/transforming growth factor β-activated kinase 1-nuclear factor-κB signals in BV-2 microglia.

    Science.gov (United States)

    Huang, Chao; Lu, Xu; Wang, Jia; Tong, Lijuan; Jiang, Bo; Zhang, Wei

    2015-08-01

    Inducible nitric oxide synthase (iNOS) critically contributes to inflammation and host defense. The inhibition of heat shock protein 70 (Hsp70) prevents iNOS induction in lipopolysaccharide (LPS)-stimulated macrophages. However, the role and mechanism of endogenous Hsp70 in iNOS induction in microglia remains unclear. This study addresses this issue in BV-2 microglia, showing that Hsp70 inhibition or knockdown prevents LPS-induced iNOS protein expression and nitric oxide production. Real-time PCR experiments showed that LPS-induced iNOS mRNA transcription was blocked by Hsp70 inhibition. Further studies revealed that the inhibition of Hsp70 attenuated LPS-stimulated nuclear translocation and phosphorylation of nuclear factor (NF)-κB as well as the degradation of inhibitor of κB (IκB)-α and phosphorylation of IκB kinase β (IKKβ). This prevention effect of Hsp70 inhibition on IKKβ-NF-κB activation was found to be dependent on the Ca(2+) /calcium-calmodulin-dependent protein kinase II (CaMKII)/transforming growth factor β-activated kinase 1 (TAK1) signals based on the following observations: 1) chelation of intracellular Ca(2+) or inhibition of CaMKII reduced LPS-induced increases in TAK1 phosphorylation and 2) Hsp70 inhibition reduced LPS-induced increases in CaMKII/TAK1 phosphorylation, intracellular pH value, [Ca(2+) ]i , and CaMKII/TAK1 association. Mechanistic studies showed that Hsp70 inhibition disrupted the association between Hsp70 and Na(+) /H(+) exchanger 1 (NHE1), which is an important exchanger responsible for Ca(2+) influx in LPS-stimulated cells. These studies demonstrate that the inhibition of endogenous Hsp70 attenuates the induction of iNOS, which likely occurs through the disruption of NHE1/Hsp70-Ca(2+) -CaMKII/TAK1-NF-κB signals in BV-2 microglia, providing further insight into the functions of Hsp70 in the CNS. PMID:25691123

  5. SPECTROSCOPIC STUDIES OF INHIBITION OF CALMODULIN ACTIVITY BY SOME DRUGS

    Directory of Open Access Journals (Sweden)

    Naderi

    1996-06-01

    Full Text Available The effect of four inhibitors on calmalulin (CuM were studied by a ftuorescence and ultraviolet techniques. Four compounds IN - ( 6 - aminohexyt 5-chloro - I - napthalenesulphonamide] (W-7, 1 - [ bis - (4 - chtorophenyt methyl] - 3 - [2, 4-dichloro - β - ( 2 , 4 - dichlorobenzyloxyl phenethyt] imidazolium chloride (R24571, trifluoperazine (TFP , thiodiphenylamide chloride (TDPAC showed inhibitory effect on bovine brain phosphodiesterase (PDE induced by CaM. The concentration of inhibitors producing 50% inhibition of of Ca 2+ / CaM activity activity (IC50 and the Hill coefficient were correlating closely between the methods, Ki's and thermodynamic parameters for these interactions were estimated.

  6. Calcium binding to calmodulin by molecular dynamics with effective polarization

    Czech Academy of Sciences Publication Activity Database

    Kohagen, Miriam; Lepšík, Martin; Jungwirth, Pavel

    2014-01-01

    Roč. 5, č. 22 (2014), s. 3964-3969. ISSN 1948-7185 R&D Projects: GA ČR GBP208/12/G016; GA MŠk LH12001 Institutional support: RVO:61388963 Keywords : EF-hand motif * free energy calculations * charge scaling * calcium-binding protein * umbrella sampling Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 7.458, year: 2014

  7. Expression of calcium/calmodulin-dependent protein kinase Ⅱ in the hippocampal subregions of the rat during postnatal development%钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠生后海马发育中的表达

    Institute of Scientific and Technical Information of China (English)

    刘伟亚; 常丽荣; 凌薇; 高香红; 宋一志; 陆涛; 武艳

    2012-01-01

    目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达.方法 应用免疫荧光方法检测CaMKⅡ在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况(n=48). 结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强,生后第10天(P10)达高峰期,此后逐渐减弱;于CA3区的表达在P4和P10时均较高.其中,CaMKⅡ在CA3区的表达高于在CA1区和DG的表达,在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达. 结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式,这可能与其在生后发育过程中的突触发生,树突、轴突形成,海马的成熟以及学习记忆功能相关.%Objective To investigate the expression of calcium/calmodulin-dependent protein kinase Ⅱ ( CaMK Ⅱ ) in the Wistar rat hippocampus during postnatal development. Methods Immunofluorescent staining was applied to observe the expression of CaMK Ⅱ in the CA1, CA3 and dentate gyms ( DG) of the rat hippocampus during postnatal development (n =48). Results In CA1 and DG, CaMK II expression increased with age, reached a plateau at postnatal day 10 ( P10) and then declined gradually. In CA3 , CaMK Ⅱexpression reached a plateau at P4 and P10. The expression of CaMK Ⅱ in CA3 was higher than that in CA1 and DG. CaMK Ⅱ expression in the stratum polymorphum and stratum moleculare was much higher than that in the stratum pyramidale or granular cell layer. Conclusion The expression of CaMK Ⅱ has a specific temporal-spatial distribution pattern. The specific temporal-spatial distribution pattern may be important to the different physiological functions of CaMK Ⅱ during postnatal development such as synaptogenesis, axonal and dendritic arborization, the maturity of hippocampus, learning and memory.

  8. 钙/钙调素依赖性蛋白激酶Ⅱ抑制剂对新生大鼠心房肌细胞钙超载的干预作用%Interventional effect of inhibitor of calcium/calmodulin-dependent protein kinaseⅡ on calcium overloading of atrial muscle cells in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    丁翔; 仝识非; 秦瑶; 宋治远

    2010-01-01

    目的 观察钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ, CaMK Ⅱ)抑制剂KN93对新生大鼠心房肌细胞钙负荷的影响,并对细胞CaMK Ⅱ的表达变化进行检测. 方法 新生大鼠心房肌细胞原代培养96 h,应用钙离子导入剂ionomycin(1.0 μmol/L)建立心房肌细胞钙超载模型,并在KN93 3种浓度(0.25、0.5、1.0 μmol/L)的干预下,以钙离子指示剂Fluo-3 /AM负载心房肌细胞,激光共聚焦显微镜观察心房肌细胞内游离钙的变化;应用Western blot法检测CaMK Ⅱ表达的变化. 结果 ①细胞培养至第4天,免疫组织化学染色90%以上细胞α-肌动蛋白抗体阳性.②与对照组比较,钙离子导入剂ionomycin明显增加细胞内钙离子荧光值[(660.16±108.47) vs (376.12±57.57),P<0.01];KN93对细胞内钙离子荧光值无明显影响(P>0.05).③预先加入3种不同浓度KN93可显著降低ionomycin导致的细胞内钙离子荧光强度的增加幅度(P<0.01),与对照组比较差异也有统计学意义(P<0.01).④钙超载组细胞CaMK Ⅱ表达较对照组明显增加(P<0.01);而KN93对细胞CaMK Ⅱ表达影响不明显.⑤不同浓度KN93预处理后,钙超载组细胞CaMK Ⅱ的表达显著降低(P<0.01). 结论 CaMK Ⅱ抑制剂KN93可降低ionomycin引发的大鼠心房肌细胞钙负荷,并下调细胞CaMK Ⅱ表达.

  9. Effect of Calmodulin (CaM) on Expression of Inducible Heat Shock Protein (HSP70) in Mice(Mus musculus) Preimplantation Embryo%钙调蛋白(CaM)对附植前小鼠胚胎热休克蛋白70(HSP70)表达的影响

    Institute of Scientific and Technical Information of China (English)

    索佳佳; 张保珍; 孙春玲; 王占美; 张双; 曹荣峰; 高善颂; 田文儒

    2013-01-01

    Heat shock protein(HSP)70 is synthesized in the preimplantation embryos of cow(Bos tarurs),ewe (Ovis aries) and mice(Mus musculus) under the conditions of heat shock in vitro,which enhances their thermotolerance to protect them from further heat damage.And Calmodulin(CaM)is the one of most important multifunctional receptor of Ca2 + and plays a key role in regulating the important gene expression in cell activities.To clarity whether the CaM participating in the expression of HSP70 in the mice embryos with heat shock and verify its mechanism,mice embryos were cultured in vitro to the blastocyst stage and then were randomly divided into control group(37℃),group treated with W7 without heat shock(37℃ +W7),heat shock group(39℃)and heat shock group treated with W7.The method of RT-PCR was used to detect the gene expression of CaM,HSP70 and heat shock factorl(HSF1),and the method of Western blot was used to detect the expression of CaM,HSP70 and HSF1.The combinations of both HSP70-CaM and HSP70-HSF1 were detected by using co-immunoprecipitation(Co-IP).The results showed that the CaM and HSP70 mRNA expression significantly increased in the mice embryos treated with 39℃ for 1 h (P<0.05).However,the CaM and HSP70 mRNA expression significantly decreased in both groups of embryo cultured at 37℃ (P<0.05) and 39℃ (P<0.01) treated with W7; W7 had no effect on the HSF1 mRNA expression in any groups of embryo.The synthesis of CaM,HSP70 and HSF1 significantly increased in the embryos treated at 39℃ for 1 h (P<0.05),and the CaM synthesis significantly decreased in the embryos cultured both at 37℃ and 39℃ and treated with W7 (P<0.05).W7 had no effect on neither HSP70 nor HSF1 synthesis in the embryos cultured at 37℃ (P>0.05) while it inhibited both HSP70 and HSF1 synthesis in the embryos cultured at 39℃ (P<0.05).The study also found that CaM combined with HSP70 and formed CaM-HSP70 complex in the mice embryos.The identification of interactions

  10. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  11. NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms

    OpenAIRE

    Love, Nick R; Pollak, Nadine; Dölle, Christian; Niere, Marc; CHEN, YaoYao; Oliveri, Paola; Amaya, Enrique; Patel, Sandip; Ziegler, Mathias

    2015-01-01

    Metabolism relies on a set of molecules that provide the chemical framework for all cellular activities. Among these molecules is NADP, a metabolite synthesized from vitamin B3 that is critical for basic metabolism, calcium signaling, and antiinflammatory processes. Despite NADP’s fundamental importance, very little is known about how animal cells regulate their NADP pool. This study shows that the enzyme NAD kinase is required for maintaining NADP levels in animals, is essential for embryoni...

  12. ARRHYTHMOGENIC CALMODULIN MUTATIONS AFFECT THE ACTIVATION AND TERMINATION OF CARDIAC RYANODINE RECEPTOR MEDIATED CA2+ RELEASE

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Chazin, Walter J.; Chen, Wayne S.R.;

    . Taken together, all CaM mutations induced an excessive fractional Ca2+ release from internal Ca2+ stores. Interestingly, we demonstrate that the presence of the RyR2 CaMBD decrease the WT CaM C-lobe apparent KD for Ca2+ binding >80 fold to 30 nM. This suggests that Ca2+ binding to the C-lobe of Ca......M would be nearly saturated at diastolic Ca2+ concentrations (~100 nM), and that the C-lobe of CaM would constitutively anchor to RyR2 in a Ca2+ bound state throughout the ECC cycle. Conversely, the N-lobe of CaM seems poised to be sensing changes in physiological Ca2+ with an apparent KD of ~800 nM in...... the presence of RyR2 CaMBD. The D95V, N97S and D129G mutations lowered the affinity of Ca2+ binding of the C-lobe of CaM, to apparent KDs of ~ 140, 150, and 4000 nM, respectively, consistent with the critical role of these residues in Ca2+ binding to the C-lobe. Thus, we suggest that these mutations...

  13. Epidermal growth factor (EGF) sensitive phosphorylation of calmodulin (CAM) in A431 cell membrane

    International Nuclear Information System (INIS)

    A431, a transformed cell line, is known to contain a high concentration of EGF receptors (EGFR). Exogenous CAM, when combined with purified membrane from A431 is strongly phosphorylated in the presence of EGF. The EGF-dependent phosphorylation of CAM did not alter the normal profile of the A431 EGFR autophosphorylation, as demonstrated by SDS-PAGE and autoradiography. In addition to its EGF dependency, the presence of divalent cations is also critical for CAM phosphorylation. The presence of a divalent cation chelator, such as EGTA, caused a complete inhibition of CAM phosphorylation, which can be reversed with cations in the following order of effectiveness: Mg++ > Mn++ > Ca++. Divalent cations also break up CAM into four co-migrating bands as indicated by Coomassie Blue stained gels and the corresponding autoradiograms. Double antibody precipitation followed by phospho-amino acid analysis revealed that the EGF-sensitive CAM phosphorylation occurs exclusively on the serine residue. Using radioimmunoassay, purified A431 membrane was shown to contain a significant amount of endogenous CAM. The implications of the EGF-sensitive CAM phosphorylation are currently under investigation

  14. Developmental expression pattern of calmodulin gene in amphioxus Branchiostoma belcheri tsingtauense

    Science.gov (United States)

    Luan, Jing; Geng, Jinpei; Fang, Shaoqing; Su, Zhiping; Zhang, Jingxuan; Xu, Hongyan; Wang, Ying; Lu, Min; Yin, Weili; Meng, Guangxiao

    2010-11-01

    We investigated the developmental expression pattern of AmphiCaM in cephalochordate amphioxus ( Branchiostoma belcheri tsingtauense). We cultured and sampled the animals at different developmental stages (eggs and larvae), and used in-situ hybridization and northern blotting to document the spatial and temporal changes in AmphiCaM expression. The alimentary tract dominates the development from the late neurula stage to the adult stage. AmphiCaM expression increased significantly in the alimentary tract during the late neurula stage and remained elevated in the adults. Our results indicate that AmphiCaM is involved in the differentiation of the alimentary tract in amphioxus; and furthermore, provide an insight into the change in function of CaM genes during evolution.

  15. Calcium/calmodulin-dependent kinase II and Alzheimer’s disease

    OpenAIRE

    Ghosh, Anshua; Giese, Karl Peter

    2015-01-01

    CaMKII is a remarkably complex protein kinase, known to have a fundamental role in synaptic plasticity and memory formation. Further, CaMKII has also been suggested to be a tau kinase. CaMKII dysregulation may therefore be a modulator of toxicity in Alzheimer’s disease, a dementia characterised by aberrant calcium signalling, synapse and neuronal loss, and impaired memory. Here, we first examine the evidence for CaMKII dysregulation in Alzheimer’s patients and draw parallels to findings in di...

  16. NRIP, a Novel Calmodulin Binding Protein, Activates Calcineurin To Dephosphorylate Human Papillomavirus E2 Protein▿

    OpenAIRE

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-01-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interferi...

  17. Role of coated vesicles, microfilaments, and calmodulin in receptor- mediated endocytosis by cultured B lymphoblastoid cells

    OpenAIRE

    1980-01-01

    Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) bindi...

  18. Hintoniamine, a new calmodulin inhibitor from the endophytic fungal species 39140-2

    Science.gov (United States)

    A new fungal endophytic species (39140-2) was isolated from the medicinal plant Hintonia latiflora (Sessé et Mociño ex DC.) Bullock, a Rubiaceae widely used in Mexico as antidiabetic agent. Sequencing of the 28S fraction of the rDNA of this fungal strain and comparing with similar sequences for all ...

  19. Calmodulin Isoforms in Plants%植物的钙调素亚型

    Institute of Scientific and Technical Information of China (English)

    刘曼; 毛国红; 孙大业

    2005-01-01

    钙调素是一种高度保守的多功能Ca2+结合蛋白,在Ca2+信号转导途径中处于中心环节.近年来的研究表明,CaM亚型在植物中普遍存在,不同的CaM亚型与靶蛋白相互作用,它们的表达特性和生物学功能存在差异.该文介绍了植物 CaM亚型的研究现状和进展.

  20. Hypoxia regulates the cAMP- and Ca2+/calmodulin signaling systems in PC12 cells.

    Science.gov (United States)

    Beitner-Johnson, D; Leibold, J; Millhorn, D E

    1998-01-01

    Hypoxic/ischemic trauma is a primary factor in the pathology of various disease states. Yet, very little is known about the molecular mechanisms involved in cellular responses and adaptations to hypoxia. As a means of identifying intracellular signaling systems that are regulated in response to hypoxia, the effects of acute and chronic hypoxia on the activity of protein kinase A (PKA) and Ca2+/CaM-dependent protein kinase II (CaMK-II) were evaluated in rat pheochromocytoma (PC12) cells. Chronic (> 6 hr), but not acute exposure to hypoxia (5% O2) significantly decreased both PKA enzyme activity and immunoreactivity compared to control levels. This effect was not due to hypoxia-induced alterations in cell number or viability. Similarly, chronic hypoxia significantly decreased CaMK-II enzyme activity and protein levels in PC12 cells. These data demonstrate that down-regulation of the cAMP and Ca2+/CaM-signaling systems is a mechanism by which PC12 cells adapt to long-term hypoxia. PMID:9439610

  1. IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

    International Nuclear Information System (INIS)

    The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions

  2. Cyclic Nucleotide-Gated Channels, Calmodulin, Adenylyl Cyclase, and Calcium/Calmodulin-Dependent Protein Kinase II Are Required for Late, but Not Early, Long-Term Memory Formation in the Honeybee

    Science.gov (United States)

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee "Apis mellifera," olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM)…

  3. Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart

    NARCIS (Netherlands)

    Scholten, A.; Preisinger, C.; Corradini, E.; Bourgonje, V.J.A.; Hennrich, M.L.; van Veen, T.A.B.; Swaminathan, P.D.; Joiner, M.L.; Vos, M.A.; Anderson, M.E.; Heck, A.J.R.

    2013-01-01

    Background The multifunctional Ca2+‐ and calmodulin‐dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful

  4. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  5. Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea, Pisum sativum L.

    NARCIS (Netherlands)

    Elzenga, JTM; Staal, M; Prins, HBA

    1997-01-01

    Pathways of signal transduction of red and blue light-dependent acidification by leaf epidermal cells were studied using epidermal strips of the Argenteum mutant of Pisum sativum. In these preparations the contribution of guard cells to the acidification is minimal. The hydroxypyridine nifedipine, a

  6. Interaction of the neuronal gap junction protein Connexin 36 with alpha Calcium/Calmodulin-dependent protein Kinase II

    OpenAIRE

    Akintürk, Sulhiye Serra

    2012-01-01

    Studien implizieren, dass Cx36 ein mutmaßlicher Partner für mehrere Proteine, einschließlich α-CaMKII ist. Im ersten Teil, um die physische Interaktion zwischen Cx36 und α-CaMKII auszuwerten, Fluoreszenz-Resonanz-Energie-Transfer (FRET)-Protokoll wurde in N2A Zellen, die mit verschiedenen Cx36 transfiziert wurden, und α-CaMKII Konstrukte angewendet. Experimente für FRET Einschätzung nach Foto Inaktivierung von Cx36-EYFP wurden mit einem 514 nm Laser in Ca2+/Ionomycin behandelt und...

  7. Structural Insights into Ca2+-Calmodulin Regulation of Plectin 1a-Integrin beta 4 Interaction in Hemidesmosomes

    Czech Academy of Sciences Publication Activity Database

    Song, J.G.; Kostan, J.; Drepper, F.; Knapp, B.; Ribeiro, E.D.; Konarev, P.V.; Grishkovskaya, I.; Wiche, G.; Gregor, Martin; Svergun, D.I.; Warscheid, B.; Djinovic-Carugo, K.

    2015-01-01

    Roč. 23, č. 3 (2015), s. 558-570. ISSN 0969-2126 R&D Projects: GA MŠk 7AMB13AT012; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : plectin * integrin * hemidesmosomes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.618, year: 2014

  8. Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations

    DEFF Research Database (Denmark)

    Shamgar, Liora; Ma, Lijuan; Schmitt, Nicole; Haitin, Yoni; Peretz, Asher; Wiener, Reuven; Hirsch, Joel; Pongs, Olaf; Attali, Bernard

    2006-01-01

    The slow IKS K+ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations...... inactivation, facilitates channel assembly, and mediates a Ca(2+)-sensitive increase of IKS-current, with a considerable Ca(2+)-dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or IKS channels with a Ca(2+)-insensitive CaM mutant markedly suppresses the currents and produces...... cannot restore normal levels of IKS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca(2+)-sensitive IKS-current stimulation, which increases the cardiac repolarization reserve and hence...

  9. Analysis of common bean (Phaseolus vulgaris L., genotype BAT93) calmodulin cDNA using computational tools

    OpenAIRE

    Kassim Amelia; Jasvin Singh; Farida Habib Shah; Subhash J Bhore

    2015-01-01

    Background: Common bean (Phaseolus vulgaris L.) is an important part of the human diet and serves as a source of natural products. Identification and understanding of genes in P. vulgaris is important for its improvement. Characterization of expressed sequence tags (ESTs) is one of the approaches in understanding the expressed genes. For the understanding of genes expression in P. vulgaris pod-tissue, research work of ESTs generation was initiated by constructing cDNA libraries using 5-day an...

  10. Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

    OpenAIRE

    Ji-Hee Kim; Ga-Young Park; Soo Young Bang; Sun Young Park; Soo-Kyung Bae; YoungHee Kim

    2014-01-01

    Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1) which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide production via downregulation of nuclear factor k...

  11. Folic Acid Reverses Nitric Oxide Synthase Uncoupling and Prevents Cardiac Dysfunction in Insulin Resistance: Role of Ca2+/Calmodulin-Activated Protein Kinase II

    OpenAIRE

    Roe, Nathan D.; He, Emily Y.; Wu, Zhenbiao; Ren, Jun

    2013-01-01

    Nitric oxide synthase (NOS) may be uncoupled to produce superoxide rather than nitric oxide (NO) under pathological conditions such as diabetes mellitus and insulin resistance, leading to cardiac contractile anomalies. Nonetheless, the role of NOS uncoupling in insulin resistance-induced cardiac dysfunction remains elusive. Given that folic acid may produce beneficial effect for cardiac insufficiency partially through its NOS recoupling capacity, this study was designed to evaluate the effect...

  12. Influence of the Calmodulin Antagonist EBB on Cyclin B1 and Cdc2-p34 in Human Drug-resistant Breast Cancer MCF-7/ADR Cells

    Institute of Scientific and Technical Information of China (English)

    Xu Shi; Huifang Zhu; Yanhong Cheng; Linglin Zou; Dongsheng Xiong; Yuan Zhou; Ming Yang; Dongmei Fan; Xiaohua Dai; Chunzheng Yang

    2008-01-01

    OBJECTIVE To investigate the influence of O-(4-ethoxyl-butyl)-berbamine (EBB) on the expression of cyclin B1 and cdc2-p34 in the human drug-resistant breast cancer MCF-7/ADR cell line.METHODS The MTT assay was used to assess the cytotoxicity of EBB. Different levels of EBB were added to different cell lines at series of time points solely or combined with doxorubicin (DOX)to detect the effect on the expression of cyclinB1 and cdc2-p34 by Western blots, cdc2-p34 tyrosine phosphorylation was detected by immunoprecipitation. In addition, apoptosis and cytoplastic Ca2+concentrations were systematically examined by laser scanning confocal microscopy (LSCM).RESULTS EBB showed little inhibitory activity on human umbilical vein endothelial cells (ECV304), whereas EBB inhibited cell growth (IC50 range, 4.55~15.74 μmol/L) in a variety of sensitive and drug-resistance cell lines. EBB also down-regulated the expression of cyclin B1 and cdc2-p34 in a concentration and time dependent manner, which was an important reason for the G2/M phase arrest. EBB was shown to induce apoptosis of MCF-7/ADR cells while increasing the level of cytoplastic Ca2+.CONCLUSION The low cytotoxicity of EBB suggests it may be useful as a rational reversal agent. The effect of EBB on cell cycle arrest and related proteins, apoptosis, and cytoplastic Ca2+ concentration may be involved in reversing multidrug resistance.

  13. Possible role of the CA2+/calmodulin dependent kinase II phosphorylation site T704 in acute desensitization of the vanilloid receptor TRPV1

    Czech Academy of Sciences Publication Activity Database

    Benedikt, Jan; Toušová, Karolina; Sušánková, Klára; Samad, Abdul; Vyklický st., Ladislav; Teisinger, Jan; Vlachová, Viktorie

    Praha : The Czech Neuroscience Society, 2007. s. 36-36. [Conference of the Czech Neuroscience Society /6./. 19.11.2007-20.11.2007, Praha] R&D Projects: GA ČR(CZ) GA305/06/0319; GA MŠk(CZ) LC554; GA MŠk(CZ) 1M0517 Institutional research plan: CEZ:AV0Z50110509 Keywords : spo2 * TRPV 1 * capsaicin Subject RIV: ED - Physiology

  14. Possible role of the ca2+/calmodulin dependent kinase II phosphorylation site T704 in acute desensitization of the vanilloid receptor TRPV1

    Czech Academy of Sciences Publication Activity Database

    Benedikt, Jan; Toušová, Karolina; Sušánková, Klára; Samad, Abdul; Vyklický st., Ladislav; Teisinger, Jan; Vlachová, Viktorie

    Breckenridge : Keystone Symposia, 2007. s. 49-49. [Keystone Symposia on Molecular and Cellular Biology. 18.09.2007-23.09.2007, Breckenridge] R&D Projects: GA ČR(CZ) GA305/06/0319; GA ČR(CZ) GA303/07/0915; GA MŠk(CZ) LC554; GA MŠk(CZ) 1M0517 Institutional research plan: CEZ:AV0Z50110509 Keywords : spo2 * capsaicin * TRPV1 Subject RIV: ED - Physiology

  15. Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth*

    OpenAIRE

    Takemura, Miyohiko; Mishima, Toshiaki; Wang, Yan; Kasahara, Jiro; Fukunaga, Kohji; Ohashi, Kazumasa; Mizuno, Kensaku

    2009-01-01

    Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppres...

  16. Die Rolle der Calcium/Calmodulin Kinase II in der Desfluran-induzierten Präkonditionierung und der Kardioprotektion durch Metoprolol

    OpenAIRE

    Schnupp, Verena

    2010-01-01

    Die Präkonditionierung ist ein endogener Schutzmechanismus, bei dem die Toleranz einer Zelle gegen die Auswirkungen eines späteren ischämischen Schadens erhöht wird. Volatile Anästhetika sind in der Lage den durch die Ischämie verursachten Gewebsschaden zu vermindern, indem sie diesen Schutzmechanismus aktivieren. Ziel der vorliegenden Arbeit war die Untersuchung der CaMK II in der Anästhetika-induzierten Präkonditionierung und in der durch Metoprolol vermittelten Kardioprotektion, sowie der ...

  17. Enzyme Dynamic Study on Calmodulin Ca++-Mg++-ATPase System of Red Blood Cells Inhibited by α-anordrin and Probimane

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    EnzymeDynamicStudyonCalmodulinCa++┐Mg++┐ATPaseSystemofRedBloodCelsInhibitedbyα┐anordrinandProbimaneLuDayongChenEnhong*CaoJing...

  18. Clozapine Functions Through the Prefrontal Cortex Serotonin 1A Receptor to Heighten Neuronal Activity via Calmodulin Kinase II-NMDA Receptor Interactions

    OpenAIRE

    Purkayastha, Sudarshana; Ford, Jason; Kanjilal, Baishali; Diallo, Souleymane; Del Rosario Inigo, Joseph; Neuwirth, Lorenz; El Idrissi, Abdelsem; Ahmed, Zaghloul; Wieraszko, Andrzej; Efrain, C.; Azmitia; Banerjee, Probal

    2011-01-01

    Aberrant dopamine release in the prefrontal cortex (PFC) is believed to underlie schizophrenia, but the mechanistic pathway through which a widely used antipsychotic, clozapine (Clz), evokes neurotransmitter-releasing electrical stimulation is unclear. We analyzed Clz-evoked regulation of neuronal activity in the PFC by stimulating axons in layers IV and V and recording the electrical effect in the postsynaptic pyramidal cells of layers II and III. We observed a Clz-evoked increase in populat...

  19. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg; Nissen, Poul

    2010-01-01

    presynaptic and postsynaptic Ca2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium......Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for...

  20. Protein (Viridiplantae): 357112535 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 368:1152 PREDICTED: putative calmodulin-like protein 6-like Brachypodium distachyon MCPGGRYAGLDIPAGAGAADLRPAFDVLDADHD...GRISREDLKSFYAKAGAHEPFDDDDIAAMIAAADADHDGFVQYDEFEGLLGRAAATGTAGGCRSAMEDAFRLMDRDGDGKVGFEDLKAYLGWAGMPVADDEIRAMIGMAGDVDGGVGLEAFARVLAVDLDGIL ...

  1. Secretion of Calmodulin in Transgenic SCaM-GFP Tobacco%转SCaM-GFP融合基因烟草中钙调素分泌特性的研究

    Institute of Scientific and Technical Information of China (English)

    周华林; 马力耕; 刘曼; 毛国红; 孙大业

    2001-01-01

    @@ 钙调素是一种重要的Ca2+结合蛋白,在细胞内发挥着重要的生物学功能.一系列实验证实,钙调素也普遍存在于动植物细胞外[1-3],并且具有重要的生物学功能[4-6].我们实验室的研究表明:胞内外钙调素在Ca2+依赖性以及在靶酶激活特性等方面基本相同,但在Ca2+亲合力等方面存在差异[2,7].近年来,在植物细胞中发现存在多种钙调素亚型,特别是在大豆中对钙调素亚型研究得比较清楚[8].通过对大豆中钙调素亚型亚细胞定位的研究,一方面可以阐明钙调素是否具有向胞外主动分泌的特性,另一方面可以确定向细胞外分泌的钙调素是否具有亚型的特异性.本文以GFP作为报告基因研究了大豆SCaM1和SCaM4的亚细胞定位分布.

  2. Untersuchung der Bedeutung der Phosphatidylinositol 3-Kinase und der Calzium/Calmodulin-abhängigen Proteinkinase II delta 2 in INS-1 Insulinom-Zellen und daraus entwickelten gentechnisch modifizierten Zellklonen für die Insulinsekretion

    OpenAIRE

    Mayer, Christian

    2001-01-01

    Für das Verständnis des Diabetes mellitus ist der Vorgang der Abgabe von Insulin ins Blut von großer Bedeutung. Dehalb wurden zwei potentielle Signalwege zur Steuerung der Insulinsekretion untersucht. Als Modell dienten Insulinom- Zellen (INS-1) und gentechnisch modifizierte Zellklone -CaMK IIdelta 2 überexpremierende INS-PAM10 und CaMK IIdelta 2 supprimierte INS-W12. Die Bestimmung der Insulinkonzentration im Überstand erfolgte durch ein RIA. Es zeigte sich keine Abh. der Insulin...

  3. CA2+/CALMODULIN-DEPENDENT KINASE II- ASSOCIATES WITH THE C TERMINUS OF THE DOPAMINE TRANSPORTER AND INCREASES AMPHETAMINE-INDUCED DOPAMINE EFFLUX VIA PHOSPHORYLATION OF N-TERMINAL SERINES

    DEFF Research Database (Denmark)

    Fog, Jacob; Khoshbouei, H; Holy, M;

    The dopamine transporter(DAT) plays a key role in clearing extracellular dopamine(DA) from the synapse. Moreover DAT is a target for the action of widely abused psychostimulants such as cocaine and amphetamine(AMPH). AMPH is a substrate for the DAT and promotes the reversal of transport and thus...... dependent kinase II- (CaMKII- ). A direct interaction between CaMKII- and DAT C-terminus was verified by the ability of C-terminal DAT GST fusion proteins to pull down both purified CaMKII- and CaMKII- from rat brain extracts. The ability of CaMKII- to exist in a complex with DAT was supported by co...

  4. The role of the LAMMER kinase Kns1 and the calcium/calmodulin-dependent kinase Cmk2 in the adaptation of Saccharomyces cerevisiae to alkaline pH stress

    OpenAIRE

    Martinez Marshall, Maria Nieves

    2013-01-01

    Die LAMMER-Kinasen sind Dual-Spezifität-Proteinkinasen, die durch das namensgebende einzigartige LAMMER-Motiv gekennzeichnet sind. Sie sind evolutionär hoch konserviert und in den meisten Eukaryonten vorhanden. Die vorliegende Arbeit stellt die erste funktionelle Charakterisierung eines bisher kaum erforschten Vertreters der LAMMER-Proteinkinase Familie Kns1 aus der Bäckerhefe dar. Phänotypische Analysen belegten eine entscheidende Rolle für Kns1 in der Regulation der Toleranz gegenüber bas...

  5. Ca2+-mediated potentiation of the swelling-induced taurine efflux from HeLa cells: On the role of calmodulin and novel protein kinase C isoforms

    DEFF Research Database (Denmark)

    Falktoft, Birgitte; Lambert, Ian H.

    2004-01-01

    The present work sets out to investigate how Ca2+ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca2+-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation...... of the release pathway. The accelerated inactivation is not observed in hypotonic Ca2+-free or high-K+ media. Addition of Ca2+-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca2+-activated K+ channels. The taurine release from control cells...... and cells exposed to Ca2+ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca2+-augmented taurine release. Exposure to Ca2+-mobilizing agonists prior to a hypotonic challenge also...

  6. 长春花钙调素基因克隆及其假基因的识别%Molecular Cloning of A Calmodulin Gene and Identification of Its Pseudogene in Catharanthus roseus

    Institute of Scientific and Technical Information of China (English)

    苏火生; 杨云; 刘宽灿; 张治礼

    2007-01-01

    以长春花[Catharanthus roseus(L.)G.Don]叶片cDNA和基因组DNA为模板,利用PCR技术扩增得到了长春花钙调素基因447 bp的全长编码cDNA序列和2个大小不同的DNA片段.序列分析表明,DNA长片段全长1 551 bp,由2个外显子和1个内含子构成,为长春花钙调素基因编码区DNA片段;DNA小片段全长447 bp,与447 bp的长春花钙调素基因cDNA核苷酸一致性高达87%,有56个碱基的差异,其中位于226 bp处的碱基A突变为T,即由AAG突变为终止密码子TAG使翻译提前终止.推测此447 bp的DNA小片段可能为长春花钙调素基因的假基因,命名为CCaMP1.

  7. EST Table: FS761276 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available Calmodulin Bound To A Calcineurin Peptide: A New Way Of Making An Old Binding Mode pdb|2F2O|B Chain B, Struc...ture Of Calmodulin Bound To A Calcineurin Peptide: A New Way Of Making An Old Binding Mode pdb|2F2P|A Chain ...A, Structure Of Calmodulin Bound To A Calcineurin Peptide: A New Way Of Making An... Old Binding Mode pdb|2F2P|B Chain B, Structure Of Calmodulin Bound To A Calcineurin Peptide: A New Way Of Making

  8. NCBI nr-aa BLAST: CBRC-DDIS-02-0170 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DDIS-02-0170 pdb|2JC6|A Chain A, Crystal Structure Of Human Calmodulin-Depende...nt Protein Kinase 1d pdb|2JC6|C Chain C, Crystal Structure Of Human Calmodulin-Dependent Protein Kinase 1d 2JC6 9e-44 41% ...

  9. SwissProt search result: AK122154 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK122154 J033145D02 (Q8CJ27) Abnormal spindle-like microcephaly-associated protein ...homolog (Calmodulin-binding protein 1) (Spindle and hydroxyurea checkpoint abnormal protein) (Calmodulin-binding protein Sha1) ASPM_MOUSE 7e-13 ...

  10. CaM kinase IIalpha mediates norepinephrine-induced translocation of cytosolic phospholipase A2 to the nuclear envelope.

    Science.gov (United States)

    Fatima, Soghra; Yaghini, Fariborz A; Ahmed, Aftab; Khandekar, Zinat; Malik, Kafait U

    2003-01-15

    Several growth factors, hormones and neurotransmitters, including norepinephrine, increase cellular calcium levels, promoting the translocation of cytosolic phospholipase A(2) to the nuclear envelope. This study was conducted to investigate the contributions of the calcium-binding protein calmodulin and of calcium-calmodulin-dependent protein kinase II to cytosolic phospholipase A(2) translocation to the nuclear envelope elicited by norepinephrine in rabbit aortic smooth-muscle cells. Norepinephrine caused cytosolic phospholipase A(2) accumulation around the nuclear envelope as determined from its immunofluorescence; cytosolic phospholipase A(2) translocation was blocked by inhibitors of calmodulin and calcium-calmodulin-dependent protein kinase II or calcium-calmodulin-dependent protein kinase IIalpha antisense oligonucleotide. Calmodulin and calcium-calmodulin-dependent protein kinase II inhibitors did not prevent cytosolic calcium increase but attenuated cytosolic phospholipase A(2) phosphorylation caused by norepinephrine or ionomycin. In vascular smooth-muscle cells reversibly permeabilized with beta-escin and treated with alkaline phosphatase, norepinephrine failed to cause cytosolic phospholipase A(2) phosphorylation and translocation to the nuclear envelope; these effects of norepinephrine were minimized by the phosphatase inhibitor okadaic acid. Recombinant cytosolic phospholipase A(2) phosphorylated by purified calcium-calmodulin-dependent protein kinase II, but not unphosphorylated or dephosphorylated cytosolic phospholipase A(2), introduced into permeabilized vascular smooth-muscle cells in the absence of calcium accumulated around the nuclear envelope. These data suggest that norepinephrine-induced translocation of cytosolic phospholipase A(2) to the nuclear envelope is mediated by its phosphorylation by calcium-calmodulin-dependent protein kinase II and that calcium alone is insufficient for cytosolic phospholipase A(2) translocation to the nuclear

  11. Dicty_cDB: Contig-U04873-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available tative calmodulin-like protein 6; 100 4e-20 DQ112161_1( DQ112161 |pid:none) Blastocladiella emersonii centri....6 1 ( AY789854 ) Clytia gracilis voucher 812IT calmodulin gene, pa... 46 1.6 1 ( DQ145721 ) Homo sapiens v-abl Abelson muri... AY542983_1( AY542983 |pid:none) Bigelowiella natans calmodulin mRN... 152 7e-36 ( P02597 ) RecName: Full...sfr*kr*wlckysrfkth infnw*kinkrrir*yvkrrknc*wpnfmlmnllellnqanhsik*gnvilknnnnnykk n...cidg818o22, 5'end,... 46 0.012 2 ( FM992691 ) Candida dubliniensis CD36 chromosome 4, complete ... 52 0.025

  12. Dicty_cDB: SSJ513 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available l*tcqsfh*klncqrsrsts ycqtms**plalw**sinwfcc*splsfr*kdeei**pkkrwsifk*lnliiii Homology vs CSM-cDNA Score E Seq...41721 |pid:none) Xenopus laevis calcium/calmodulin-... 153 3e-36 ( Q63450 ) RecName: Full=Calcium/calmodulin...-dependent protein kina... 152 4e-36 ( Q14012 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 1...yostelium discoideum chromoso... 168 6e-41 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-Ca...SS (Link to library) SSJ513 (Link to dictyBase) - - - Contig-U11996-1 SSJ513Z (Link to Original

  13. Dicty_cDB: SFJ553 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available assxplii*knfnr*l*fprtrmdscl*tcqsfhxklncqrsrxtsycqxms**psalw* *sinwfcc*spxsxx*kdxxi* Homology vs CSM-cDNA Sco...cium/calmodulin-... 144 2e-41 (Q8IU85) RecName: Full=Calcium/calmodulin-dependent pr...otein kina... 142 2e-41 (Q8BW96) RecName: Full=Calcium/calmodulin-dependent protein kina... 142 2e-41 protei...GC82022 protein, m... 141 3e-42 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-Ca...SF (Link to library) SFJ553 (Link to dictyBase) - - - Contig-U11996-1 SFJ553Z (Link to Original

  14. Mechanism of the induction of endoplasmic reticulum stress by the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT): Activation of PERK/eIF2α, IRE1α, ATF6 and calmodulin kinase.

    Science.gov (United States)

    Merlot, Angelica M; Shafie, Nurul H; Yu, Yu; Richardson, Vera; Jansson, Patric J; Sahni, Sumit; Lane, Darius J R; Kovacevic, Zaklina; Kalinowski, Danuta S; Richardson, Des R

    2016-06-01

    The endoplasmic reticulum (ER) plays a major role in the synthesis, maturation and folding of proteins and is a critical calcium (Ca(2+)) reservoir. Cellular stresses lead to an overwhelming accumulation of misfolded proteins in the ER, leading to ER stress and the activation of the unfolded protein response (UPR). In the stressful tumor microenvironment, the UPR maintains ER homeostasis and enables tumor survival. Thus, a novel strategy for cancer therapeutics is to overcome chronically activated ER stress by triggering pro-apoptotic pathways of the UPR. Considering this, the mechanisms by which the novel anti-cancer agent, Dp44mT, can target the ER stress response pathways were investigated in multiple cell-types. Our results demonstrate that the cytotoxic chelator, Dp44mT, which forms redox-active metal complexes, significantly: (1) increased ER stress-associated pro-apoptotic signaling molecules (i.e., p-eIF2α, ATF4, CHOP); (2) increased IRE1α phosphorylation (p-IRE1α) and XBP1 mRNA splicing; (3) reduced expression of ER stress-associated cell survival signaling molecules (e.g., XBP1s and p58(IPK)); (4) increased cleavage of the transcription factor, ATF6, which enhances expression of its downstream targets (i.e., CHOP and BiP); and (5) increased phosphorylation of CaMKII that induces apoptosis. In contrast to Dp44mT, the iron chelator, DFO, which forms redox-inactive iron complexes, did not affect BiP, p-IRE1α, XBP1 or p58(IPK) levels. This study highlights the ability of a novel cancer therapeutic (i.e., Dp44mT) to target the pro-apoptotic functions of the UPR via cellular metal sequestration and redox stress. Assessment of ER stress-mediated apoptosis is fundamental to the understanding of the pharmacology of chelation for cancer treatment. PMID:27059255

  15. Polyphasic taxonomy of Aspergillus fumigatus and related species

    DEFF Research Database (Denmark)

    Hong, S.B.; Go, S.J.; Shin, H.D.;

    2005-01-01

    The variability within Aspergillus fumigalus Fresenius and related species was examined using macro-, micro-morphology, growth temperature regimes and extrolite patterns. In addition, DNA analyses including partial beta-tubulin, calmodulin and actin gene sequences were used. Detailed examination ...

  16. Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro.

    Science.gov (United States)

    Hayakawa, K; Okagaki, T; Ye, L H; Samizo, K; Higashi-Fujime, S; Takagi, T; Kohama, K

    1999-05-01

    In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles. PMID:10231551

  17. AcEST: BP914427 [AcEST

    Lifescience Database Archive (English)

    Full Text Available sp_hit_id Q6RET7 Definition sp|Q6RET7|CCAMK_MEDTR Calcium and calcium/calmodulin-depende...nt serine/threonine-protein kinase DMI-3 OS=Medicago truncatula Align length 104 Score (bit) 139.0 E-value 5.0e-33 Repo...Value sp|Q6RET7|CCAMK_MEDTR Calcium and calcium/calmodulin-dependent s... 139 5e-33 sp|Q43531|CCAMK_LILLO Ca...lcium and calcium/calmodulin-dependent s... 138 1e-32 sp|Q6RET6|CCAMK_PEA Calcium and calcium/calmodulin-depende...nt ser... 136 5e-32 sp|Q28IM6|HPCL1_XENTR Hippocalcin-like protein 1 OS=Xenop

  18. Protein (Cyanobacteria): 352974 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ociation-domain protein Synechococcus sp. CB0101 MALSDRDQEILSINQAMLDSVVNGDWSRYATFCA...ZP_07974427.1 1117:14446 1118:14646 1129:7054 232348:1931 Calcium/calmodulin dependent protein kinase II ass

  19. Protein (Cyanobacteria): 96882 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ent protein kinase II, association-domain Arthrospira sp. PCC 8005 MIKKLFCSTLSASFLAATMSGCAPVAETGEVACAEVTEAEI...ZP_09781165.1 1117:1835 1150:12908 35823:2083 376219:1678 Calcium/calmodulin depend

  20. Protein (Cyanobacteria): 352975 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ciation-domain protein Synechococcus sp. CB0205 MAFSERDQEILRLNQAMLNSVASGDWQAYSAVCAD...ZP_07970261.1 1117:14446 1118:14646 1129:7054 232363:890 Calcium/calmodulin dependent protein kinase II asso

  1. EDF-1 downregulates the CaM/Cn/NFAT signaling pathway during adipogenesis

    Energy Technology Data Exchange (ETDEWEB)

    López-Victorio, Carlos J.; Velez-delValle, Cristina; Beltrán-Langarica, Alicia [Department of Cell Biology, Center for Research and Advanced Studies-IPN, Apdo. Postal 14-740, México City 07000 (Mexico); Kuri-Harcuch, Walid, E-mail: walidkuri@gmail.com [Department of Cell Biology, Center for Research and Advanced Studies-IPN, Apdo. Postal 14-740, México City 07000 (Mexico)

    2013-03-01

    Highlights: ► EDF-1 participates early adipogenesis in 3T3F442A cells induced with Staurosporine/Dexamethasone. ► EDF-1 associates with CaM and Cn, most likely inactivating Cn. ► EDF-1/CaM complex seems to prevent NFATc1 activation by Cn. ► EDF-1 regulates the Cn/CaM/NFATc1 pathway during adipogenesis. ► EDF-1 may regulate the activation of Cn through a complex formation with CaM. - Abstract: The endothelial differentiation factor-1 (EDF-1) is a calmodulin binding protein that regulates calmodulin-dependent enzymes. In endothelial cells, this factor can form a protein complex with calmodulin. We analyzed the relationship between this factor and the members of calmodulin/calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway during adipogenesis of 3T3-F442A cells. We found that the expression of edf1 is upregulated during early adipogenesis, whereas that of calcineurin gene is lowered, suggesting that this pathway should be downregulated to allow for adipogenesis to occur. We also found that EDF-1 associates with calmodulin and calcineurin, most likely inactivating calcineurin. Our results showed that EDF-1 inactivates the calmodulin/calcineurin/NFAT pathway via sequestration of calmodulin, during early adipogenesis, and we propose a mechanism that negatively regulates the activation of calcineurin through a complex formation between EDF-1 and calmodulin. This finding raises the possibility that modulating this pathway might offer some alternatives to regulate adipose biology.

  2. Calcium microdomains near R-type calcium channels control the induction of presynaptic LTP at parallel fiber to Purkinje cell synapses

    OpenAIRE

    Myoga, Michael H.; Regehr, Wade G.

    2011-01-01

    R-type calcium channels in postsynaptic spines signal through functional calcium microdomains to regulate a calcium-calmodulin sensitive potassium channel that in turn regulates postsynaptic hippocampal LTP. Here we ask whether R-type calcium channels in presynaptic terminals also signal through calcium microdomains to control presynaptic LTP. We focus on presynaptic LTP at parallel fiber to Purkinje cell synapses in the cerebellum (PF-LTP), which is mediated by calcium/calmodulin-stimulated ...

  3. Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism

    International Nuclear Information System (INIS)

    We have shown that melatonin immediately and transiently stimulates intracellular free radical production on a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and are accompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation of AA; inhibition of Ca2+-independent, but not Ca2+-dependent, phospholipase A2 (PLA2), prevents both melatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS, indicating that PLA2 is upstream with respect to LOX, as occurs in many signaling pathways. Chlorpromazine, an inhibitor of melatonin-calmodulin interaction, inhibits both ROS and arachidonic acid production, thus possibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it is known that Ca2+-independent PLA2 binds to calmodulin: our results are compatible with PLA2 being liberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signal transduction. These results delineate a novel molecular pathway through which melatonin may participate to the inflammatory response.

  4. Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

    Science.gov (United States)

    Batters, Christopher; Woodall, Katy A; Toseland, Christopher P; Hundschell, Christian; Veigel, Claudia

    2012-08-10

    The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle. PMID:22718767

  5. Dicty_cDB: Contig-U13844-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 71757 |pid:none) E.tenella mRNA for calmodulin-domain p... 53 2e-05 CT005269_12( CT005269 |pid:none) Leishmania ma...5_23( FN357295 |pid:none) Schistosoma mansoni genome sequen... 50 1e-04 ( Q14012 ) RecName: Full=Calcium/calm...0_32( FN357490 |pid:none) Schistosoma mansoni genome sequen... 57 1e-06 AB000796_1( AB000796 |pid:none) Arabidopsis thalian... mRNA for calmodulin-domain pr... 56 3e-06 BX537313_6( BX537313 |pid:none) Zebrafish DN...um KMLC mRNA fo... 54 1e-05 DQ205646_1( DQ205646 |pid:none) Toxoplasma gondii calmodulin-like ... 54 1e

  6. Dicty_cDB: Contig-U10256-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2 1e-14 AB072805_1( AB072805 |pid:none) Decapterus maruadsi mlc2 mRNA for ... 82 ...t WO20040700... 82 2e-14 AB072810_1( AB072810 |pid:none) Decapterus tabl mlc2 mRNA for myos... 82 2e-14 (Q9L...core E Sequences producing significant alignments: (bits) Value N ( C89656 ) Dictyostelium discoideum slug c...lium discoideum slug cDNA, clone SSG876. 961 0.0 2 ( AC116979 ) Dictyostelium discoideum chromosome 2 map...id:none) Synthetic construct calmodulin mut... 113 6e-24 NRL( 1OSA ) calmodulin - Paramecium tetraurelia 113

  7. New and revisited species in Aspergillus section Nigri

    DEFF Research Database (Denmark)

    Varga, J.; Frisvad, Jens Christian; Kocsube, S.;

    2011-01-01

    based on either beta-tubulin or calmodulin sequence data. Aspergillus eucalypticola produced pyranonigrin A, funalenone, aurasperone B and other naphtho-gamma-pyrones. Aspergillus neoniger is also a biseriate species isolated from desert sand in Namibia, and mangrove water in Venezuela, which produces...... and A. awamori are also detailed. Although these species differ in their occurrence and several physiological means (elastase activities, abilities to utilise 2-deoxy-D-glucose as sole carbon source), our data indicate that only molecular approaches including sequence analysis of calmodulin or beta...

  8. Free-solution, label-free molecular interactions studied by back-scattering interferometry

    DEFF Research Database (Denmark)

    Bornhop, D.J.; Latham, J.C.; Kussrow, A.;

    2007-01-01

    Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and......, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200...

  9. Dicty_cDB: SLD285 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available cl*tcqsfh*klncqrsrstsycq tms**plalw**sinwfcc*splsfr*kdeei**pkkrwsifk*lnliiiiikkkk...modulin-... 153 8e-36 ( Q63450 ) RecName: Full=Calcium/calmodulin-dependent protein ki...na... 152 1e-35 ( Q14012 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 152 1e-35 PDBF( 1A06 )...) Dictyostelium discoideum chromoso... 163 6e-39 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-CaM-K...SL (Link to library) SLD285 (Link to dictyBase) - - - Contig-U11996-1 SLD285P (Link to Original

  10. Dicty_cDB: SLF532 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available nvilpnnvlm tfgslvvinqlvlllistlislkr*rnimtkeevvnlqiikfnnnnn Frame B: ---tilcfisklii*knfnr*l*fprtrmdscl*tcqsfh...evis MGC82022 protein, m... 74 1e-12 ( Q63450 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 7...4 2e-12 ( Q14012 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 74 ...B076903 |pid:none) Ciona intestinalis Ci-CaM-K mRNA f... 81 1e-14 BC074183_1( BC074183 |pid:none) Xenopus la...SL (Link to library) SLF532 (Link to dictyBase) - - - Contig-U11996-1 SLF532Z (Link to Original

  11. Dicty_cDB: SSH378 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available wfpti lcfisklii*knfnr*l*fprtrmdscl*tcqsfh*klncqrsrstsycqtms**plalw **sinwfcc*splsfr*kdeei**pkkrwsifk*lnliiii...M-K mRNA f... 142 5e-33 ( Q63450 ) RecName: Full=Calcium/calmodulin-dep...endent protein kina... 136 2e-31 ( Q14012 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 136 2...982 |pid:none) Dictyostelium discoideum chromoso... 147 2e-34 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-Ca...SS (Link to library) SSH378 (Link to dictyBase) - - - Contig-U11996-1 SSH378Z (Link to Original

  12. Dicty_cDB: SLH770 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ilcfisklii*knfnr*l*fprtrmdscl*tcqsfh*klncqrsrstsycqtm s**plalw**sinwfcc*splsfr*kd...74183 |pid:none) Xenopus laevis MGC82022 protein, m... 81 1e-14 ( Q63450 ) RecName: Full=Calcium/calmodulin-...dependent protein kina... 80 2e-14 ( Q14012 ) RecName: Full=Calcium/calmodulin-de...... 209 2e-53 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-CaM-K mRNA f... 87 2e-16 BC074183_1( BC0...SL (Link to library) SLH770 (Link to dictyBase) - - - Contig-U11996-1 SLH770Z (Link to Original

  13. Dicty_cDB: SLC252 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available i*knfnr*l* fprtrmdscl*tcqsfh*klncqrsrstsycqtms**plalw**sinwfcc*splsfr*k deei**pkkrwsifk*lnliiii Homology vs ...Mus musculus 13 days embryo lung c... 176 5e-43 (Q8IU85) RecName: Full=Calcium/calmodulin-dependent protein ...kina... 176 5e-43 (Q8BW96) RecName: Full=Calcium/calmodulin-dependent protein kin...none) Dictyostelium discoideum chromoso... 192 6e-48 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-Ca...SL (Link to library) SLC252 (Link to dictyBase) - - - Contig-U11996-1 SLC252Z (Link to Original

  14. Febrile infection-related status epilepticus in a child after a common infection

    DEFF Research Database (Denmark)

    Andersen, Anne Helene; Hansen, Lars Kjærsgaard

    2014-01-01

    A 13-year-old boy developed seizures and intractable status epilepticus a week after having had a sore throat. Ketogenic diet possibly had some effect. Antibodies to calmodulin dependent protein kinase II were found and could possibly suggest an immunologic aetiology.......A 13-year-old boy developed seizures and intractable status epilepticus a week after having had a sore throat. Ketogenic diet possibly had some effect. Antibodies to calmodulin dependent protein kinase II were found and could possibly suggest an immunologic aetiology....

  15. Autophosphorylation of [alpha]CaMKII is Differentially Involved in New Learning and Unlearning Mechanisms of Memory Extinction

    Science.gov (United States)

    Kimura, Ryoichi; Silva, Alcino J.; Ohno, Masuo

    2008-01-01

    Accumulating evidence indicates the key role of [alpha]-calcium/calmodulin-dependent protein kinase II ([alpha]CaMKII) in synaptic plasticity and learning, but it remains unclear how this kinase participates in the processing of memory extinction. Here, we investigated the mechanism by which [alpha]CaMKII may mediate extinction by using…

  16. Dicty_cDB: VSI369 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available VS (Link to library) VSI369 (Link to dictyBase) - - - Contig-U10256-1 VSI369F (Link to Original ... NA f... 119 5e-26 MCSFCU( JK0011 )calmodulin - sea cucumber ... (Stichopus japonicus) 119 5e-26 ( P21251 ) RecName ...

  17. Dicty_cDB: VSI464 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available VS (Link to library) VSI464 (Link to dictyBase) - - - Contig-U10256-1 VSI464F (Link to Original ... NA f... 104 1e-21 MCSFCU( JK0011 )calmodulin - sea cucumber ... (Stichopus japonicus) 104 1e-21 AY893101_1( AY8931 ...

  18. Taxonomy of Penicillium citrinum and related species

    DEFF Research Database (Denmark)

    Houbraken, J.A.M.P.; Frisvad, Jens Christian; Samson, A.F.

    2010-01-01

    Penicillium citrinum and related species have been examined using a combination of partial beta-tubulin, calmodulin and ITS sequence data, extrolite patterns and phenotypic characters. It is concluded that seven species belong to the series Citrina. Penicillium sizovae and Penicillium steckii are...

  19. Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1998-01-01

    of Schwann cells. Removal of extracellular Ca2+ by addition of EGTA to the culture medium suppressed [3H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca2+ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [3H] thymidine...

  20. New species in Aspergillus section Terrei

    DEFF Research Database (Denmark)

    Samson, R. A.; Peterson, S. W.; Frisvad, Jens Christian;

    2011-01-01

    Section Terrei of Aspergillus was studied using a polyphasic approach including sequence analysis of parts of the beta-tubulin and calmodulin genes and the ITS region, macro- and micromorphological analyses and examination of extrolite profiles to describe three new species in this section. Based...

  1. Activation of calcineurin by phosphotidylserine containing vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Politino, M.; King, M.M.

    1986-05-01

    Calcineurin (CaN) is a Ca/sup 2 +/- and calmodulin-regulated phosphatase. Recent findings suggested an association of CaN with biological membranes and prompted the present investigation into the interactions of the phosphatase with phospholipids in vitro. In the absence of calmodulin, sonicated preparations of phosphatidylserine (PS) provided a five-fold activation of the Ni- and Mn-supported activities of CaN towards (/sup 32/P) histone Hl; activation in the presence of calmodulin was much less pronounced. Half-maximal activation in the absence of calmodulin required approximately 0.1 mg/ml of PS. Activation of CaN was also observed with mixed vesicles of phosphatidylcholine (PC) containing 20% PS but not with PC alone, or with phosphatidylethanolamine (PE). Molecular sieve chromatography on Ultrogel AcA 34 provided further evidence that CaN associates with phospholipid vesicles composed of PS, or PC containing 20% PS, but not with vesicles of PC or PE. Complete association with medium sized vesicles of PS and PC/PS required Ca/sup 2 +/ ions; in the absence of the metal ion at least 60% of the enzyme failed to interact with the lipids while the remainder preferentially migrated with larger vesicles. These results suggest a role for Ca/sup 2 +/ in regulating CaN's interaction with phospholipids.

  2. Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin'

    DEFF Research Database (Denmark)

    Taniwaki, Marta Hiromi; Pitt, John I; Iamanaka, Beatriz T.;

    2015-01-01

    A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, β-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as...

  3. Four new species of Emericella from the Mediterranean region of Europe

    DEFF Research Database (Denmark)

    Zalar, Polona; Frisvad, Jens Christian; Gunde-Cimerman, Nina; Varga, János; Samson, Robert A.

    2008-01-01

    Four new species of Emericella, E. discophora, E. E. olivicola. and E. stella-maris, are proposed. Their new taxonomic status was determined applying a polyphasic taxonomic approach using phenotypic (morphology and extrolite profiles) and molecular (sequences of ITS, P-tubulin and calmodulin gene......, varitriols arid aflatoxin B-1, of which the latter was detected only in one of the two strains....

  4. Gene : CBRC-DDIS-02-0170 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DDIS-02-0170 Novel 2 C UNKNOWN KCC1D_MOUSE 6e-45 40% ref|XP_643776.1| putative protein seri ... 52341.1| similar to Xenopus laevis (African clawed frog ). Calcium/calmodulin-dependent protein kinase I al ...

  5. Domain Modeling: NP_001211.3 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_001211.3 chr7 STRUCTURE OF THE OLIGOMERISATION DOMAIN OF CALCIUM-CALMODULIN DEPE...NDENT PROTEIN KINASE II GAMMA c2ux0f_ chr7/NP_001211.3/NP_001211.3_apo_534-659.pdb blast 564G,566T,568F,571E

  6. Aspects of the regulator and physical-chemical properties of phenylalanine ammoniacliase of the fruit tissue of citrus sinensis

    International Nuclear Information System (INIS)

    The occurence of PAL as an enzyme containing calmodulin as an integral part of its structure is discussed against the background of the important regulatory function of the enzyme in secondary plant metabolism. This study also forms the basis of the investigation into the effect of gamma radiation on citrus tissue which leads to the induction of phenylalanineammoniacliase

  7. The Nuclear Transcription Factor RAR Associates with Neuronal RNA Granules and Suppresses Translation

    Science.gov (United States)

    All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II alpha and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionat...

  8. Two-photon polarization microscopy reveals protein structure and function

    Czech Academy of Sciences Publication Activity Database

    Lazar, Josef; Bondar, Alexey; Timr, S.; Firestein, S. J.

    2011-01-01

    Roč. 8, č. 8 (2011), s. 684-U120. ISSN 1548-7091 Institutional research plan: CEZ:AV0Z60870520 Keywords : green fluorescent protein s * living cells * in-vivo * indicators * anisotropy * activacion * dissociation * orientation * calmodulin * membranes Subject RIV: CE - Biochemistry Impact factor: 19.276, year: 2011

  9. A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions.

    Science.gov (United States)

    Herling, Therese W; O'Connell, David J; Bauer, Mikael C; Persson, Jonas; Weininger, Ulrich; Knowles, Tuomas P J; Linse, Sara

    2016-05-10

    The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions. PMID:27166804

  10. Cholinergic Neurons Mediate CaMKII-Dependent Enhancement of Courtship Suppression

    Science.gov (United States)

    Mehren, Jennifer E.; Griffith, Leslie C.

    2006-01-01

    In "Drosophila," calcium/calmodulin-dependent protein kinase II (CaMKII) activity is crucial in associative courtship conditioning for both memory formation and suppression of courtship during training with a mated female. We have previously shown that increasing levels of constitutively active CaMKII, but not calcium-dependent CaMKII, in a subset…

  11. Taxonomic studies of the Penicillium glabrum complex and the description of a new species P. subericola

    DEFF Research Database (Denmark)

    Barreto, M. C.; Houbraken, J.; Samson, R. A.;

    2011-01-01

    of P. spinulosum and a third group contained isolates which were unique in their β-tubulin and calmodulin sequences, extrolite profiles and growth characteristics. This group of isolates is described as the new species Penicillium subericola. The type strain of P. subericola CBS 125096T was isolated...... from Portuguese raw cork, but additional isolates were found from soil, air and lumen....

  12. Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea

    NARCIS (Netherlands)

    Hayashi, K.; Schoonbeek, H.; Waard, de M.A.

    2003-01-01

    Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin anta

  13. EST data: 6606 [RED

    Lifescience Database Archive (English)

    Full Text Available RA3289 D39297 AU032040 DPlate 068 F10 5' CCA TCC GCT ACG ACG ACT TC 3' 5' TCA CAC CGG AAT GAA AA ... C AC 3' >CHKCM11_1(K00510|pid:g211542) Chicken ... cCM1 gene coding for a calmodulin-like protein; ca ...

  14. Polyphasic taxonomy of Aspergillus fumigatus and related species

    DEFF Research Database (Denmark)

    Hong, S.B.; Go, S.J.; Shin, H.D.; Frisvad, Jens Christian; Samson, R.A.

    2005-01-01

    the A. fumigatus sensu stricto species. A. lentulus including isolates from clinical origin, Korean soil and from a dolphin Clustered into an isolated group based on beta-tubulin, calmodulin and actin gene sequences, differing from A. fumigalus by morphological characters, growth temperature and...

  15. Delimitation and characterisation of Talaromyces purpurogenus and related species

    DEFF Research Database (Denmark)

    Yilmaz, N.; Houbraken, J.; Hoekstra, E. S.;

    2012-01-01

    Taxa of the Talaromyces purpurogenus complex were studied using a polyphasic approach. ITS barcodes were used to show relationships between species of the T. purpurogenus complex and other Talaromyces species. RPB1, RPB2, beta-tubulin and calmodulin sequences were used to delimit phylogenetic...

  16. (1)H, (13)C and (15)N NMR assignments of a calcium-binding protein from Entamoeba histolytica.

    Science.gov (United States)

    Verma, Deepshikha; Bhattacharya, Alok; Chary, Kandala V R

    2016-04-01

    We report almost complete sequence specific (1)H, (13)C and (15)N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization. PMID:26377206

  17. Characterization of transport of calcium by microsomal membranes from roots maize

    Energy Technology Data Exchange (ETDEWEB)

    Vaughan, M.A.

    1985-01-01

    This study investigates calcium transport by membranes of roots of maize isolated by differential centrifugation. The preparation was determined to be enriched in plasma membrane using market enzyme and electron microscopy. Using the /sup 45/Ca filtration technique and liquid scintillation counting, vesicular calcium uptake was shown to be stimulated by added calmodulin and specific for and dependent on ATP. Conditions for maximal calcium accumulation were found to be 30 min incubation in the presence of 5 mM ATP, 5 mM MgCl/sub 2/, 50 ..mu..M CaCl/sub 2/, at 23/sup 0/C, and at pH 6.5. Calcium uptake was inhibited by the ionophores A23187, X-537A, and ionomycin. Sodium fluoride, ruthenium red, and p-chloromercuribenzoate completely inhibited transport: diamide and vanadate produced slight inhibition; caffeine, caffeic acid, oligomycin, and ouabain produced little or no inhibition. Chlorpromazine, W7, trifluoperazine, and R 24 571 inhibit calcium uptake irrespective of added calmodulin, while W5 showed little effect on uptake. Verapamil, nifedipine, cinnarizine, flunarizine, lidoflazine, and diltiazem decreased calcium uptake by 17%-50%. Electron microscopic localization of calcium by pyroantimonate showed vesicles incubated with calmodulin and ATP showed the greatest amount of precipitate. These results suggest that these vesicles accumulate calcium in an ATP-dependent, calmodulin-stimulated manner.

  18. GenBank blastx search result: AK058494 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058494 001-016-E06 X04259.1 Caenorhabditis elegans calmodulin-like gene cal-1 located 52-68% f ... rom the left end of chromosome IV (see the Salvato paper ).|INV INV 3e-21 +1 ...

  19. GenBank blastx search result: AK119167 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119167 001-036-F05 X04259.1 Caenorhabditis elegans calmodulin-like gene cal-1 located 52-68% f ... rom the left end of chromosome IV (see the Salvato paper ).|INV INV 5e-48 +3 ...

  20. GenBank blastx search result: AK058646 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058646 001-018-E09 X04259.1 Caenorhabditis elegans calmodulin-like gene cal-1 located 52-68% f ... rom the left end of chromosome IV (see the Salvato paper ).|INV INV 2e-47 +1 ...

  1. GenBank blastx search result: AK059534 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059534 001-029-D11 X04259.1 Caenorhabditis elegans calmodulin-like gene cal-1 located 52-68% f ... rom the left end of chromosome IV (see the Salvato paper ).|INV INV 6e-48 +1 ...

  2. The promise of CaMKII inhibition for heart disease : preventing heart failure and arrhythmias

    NARCIS (Netherlands)

    Westenbrink, B. Daan; Edwards, Andrew G.; McCulloch, Andrew D.; Brown, Joan Heller

    2013-01-01

    Introduction: Calcium-calmodulin-dependent protein kinase II (CaMKII) has emerged as a central mediator of cardiac stress responses which may serve several critical roles in the regulation of cardiac rhythm, cardiac contractility and growth. Sustained and excessive activation of CaMKII during cardia

  3. Connecting RNA Processing to Abiotic Environmental Response in Arabidopsis: the role of a polyadenylation factor

    Science.gov (United States)

    Li, Q. Q.; Xu, R.; Hunt, A. G.; Falcone, D. L.

    Plants are constantly challenged by numerous environmental stresses both biotic and abiotic It is clear that plants have evolved to counter these stresses using all but limited means We recently discovered the potential role of a messenger RNA processing factor namely the Arabidopsis cleavage and polyadenylation specificity factor 30 kDa subunit AtCPSF30 when a mutant deficient in this factor displayed altered responses to an array of abiotic stresses This AtCPSF30 mutant named oxt6 exhibited an elevated tolerance to oxidative stress Microarray experiments of oxt6 and its complemented lines revealed an altered gene expression profile among which were antioxidative defense genes Interestingly the same gene encoding AtCPSF30 can also be transcribed into a large transcript that codes for a potential splicing factor Both protein products have a domain for RNA binding and a calmodulin binding domain activities of which have been confirmed by biochemical assays Surprisingly binding of AtCPSF30 to calmodulin inhibits the RNA-binding activity of the protein Mutational analysis shows that a small part of the protein is responsible for calmodulin binding and point mutations in this region abolished both RNA binding activity and the inhibition of this activity by calmodulin Analyses of the potential splicing factor are on going and the results will be presented The interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through

  4. Sex-Dependent Up-Regulation of Two Splicing Factors, Psf and Srp20, during Hippocampal Memory Formation

    Science.gov (United States)

    Antunes-Martins, Ana; Mizuno, Keiko; Irvine, Elaine E.; Lepicard, Eve M.; Giese, K. Peter

    2007-01-01

    Gene transcription is required for long-term memory (LTM) formation. LTM formation is impaired in a male-specific manner in mice lacking either of the two Ca[superscript 2+] / calmodulin-dependent kinase kinase ("Camkk") genes. Since altered transcription was suggested to cause these impairments in LTM formation, we used microarrays to screen for…

  5. Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast

    DEFF Research Database (Denmark)

    Kleinow, T.; Bhalerao, R.; Breuer, F.; Umeda, M.; Salchert, Klaus-Dieter; Koncz, C.

    2000-01-01

    , zinc-finger factors AZF2 and ZAT10, as well as orthologs of hexose/UDP-hexose transporters, calmodulin, SMC1-cohesin and Snf4. Here we describe the characterization of AtSNF4, a functional Arabidopsis Snf4 ortholog, that interacts with yeast Snf1 and specifically binds to the C-terminal regulatory...

  6. Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

    Science.gov (United States)

    Luo, Mingzhi; Yang, Zhouqi; Li, Jingbao; Xu, Huiyun; Li, Shengsheng; Zhang, Wei; Qian, Airong; Shang, Peng

    2013-06-01

    We have explored the role of Ca2+ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca2+]i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca2+]i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca2+]i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca2+ influx through stretch-activated channels, then results in microfilament disruption.

  7. Dicty_cDB: Contig-U14093-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e) Homo sapiens BAC clone RP11-83F13 ... 46 0.001 MCSP( A03024 )calmodulin - spinach (tentative seque...WO200711... 65 2e-09 AM437552_1( AM437552 |pid:none) Vitis vinifera contig VV78X030840.... 65 2e-09 ( P28582 ) RecName: Full=Calci...e) Mus musculus skin calmodulin-relat... 55 3e-06 AY484589_7( AY484589 |pid:none) Musa acuminata clone MuH9, genomi... 2e-05 M84155_1( M84155 |pid:none) Entamoeba histolytica calcium-binding ... 52 2... fast skeletal muscle t... 51 3e-05 AJ344156_1( AJ344156 |pid:none) Nicotiana benthamiana

  8. Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata.

    Science.gov (United States)

    Holzinger, Andreas; Lütz-Meindl, Ursula

    2002-01-01

    The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus. PMID:12175672

  9. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    International Nuclear Information System (INIS)

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (32P) PO43- and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone

  10. Dicty_cDB: Contig-U14752-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 70 3e-11 AY542983_1( AY542983 |pid:none) Bigelowiella natans calmodulin mRN... 70 3e-11 ( P11120 ) RecName: Full...ne A018 cal... 62 8e-09 S58314( S58314 )calmodulin - moss (Physcomitrella patens) 62 8e-09 DQ916806_1( DQ916806 |pid:none) Galdieri... ) CAWT18478.rev CAWT Capitella sp. I ECS-2004 Prima... 42 0.038 2 ( EY550439 ) CAWT18478.fwd CAWT Capitella sp. I ECS-2004 Pri...ma... 42 0.039 2 ( EY537428 ) CAWT10468.rev CAWT Capitella sp. I ECS-2004 Prima... 42 0.0...40 2 ( EY537429 ) CAWT10468.fwd CAWT Capitella sp. I ECS-2004 Prima... 42 0.041 2 ( EF533700 ) Glycine max c

  11. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

    DEFF Research Database (Denmark)

    Peters, C; Bayer, M J; Bühler, S;

    2001-01-01

    SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodu......SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2......+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans...

  12. Differential effects of protein kinase inhibitors and activators on memory formation in the 2-day-old chick.

    Science.gov (United States)

    Serrano, P A; Beniston, D S; Oxonian, M G; Rodriguez, W A; Rosenzweig, M R; Bennett, E L

    1994-01-01

    Thirteen protein kinase inhibitors (PKIs) were investigated in chicks for their in vitro effects on PKC activity and for their in vivo effects on memory formation for a peak-avoidance task. Amnesia occurred by 15-30 min post-training when agents that inhibit primarily Ca2+/calmodulin were injected into brain. Amnesia occurred by 60 min post-training when agents that inhibit PKC-, PKA-, and/or PKG-dependent protein kinases, but not Ca2+/calmodulin, were injected. Enhancement of memory formation was accomplished by injecting bradykinin, but not forskolin. Both of these agents, however, attenuated the amnesia produced by H-7. These results are discussed as relevant neural processes involved in memory and synaptic plasticity. PMID:8129687

  13. Dicty_cDB: SSM811 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 054 chromoso... 90 3e-17 DQ122882_1( DQ122882 |pid:none) Chlamydomonas incerta calmodulin m... 86 3e-16 ( P04352 ) RecName: Full=Cal...|C97784.1 Oryza sativa cDNA, partial sequence (C62862_2A). 32 1.5 2 AL050329 |AL0...um myosin calcium-b... 167 1e-40 CR382133_531( CR382133 |pid:none) Debaryomyces hanse...modulin; Short=CaM; &M20729_... 86 3e-16 NRL( 1CLM ) calmodulin - Paramecium tetraureli... 12.0 %: cytoskeletal 4.0 %: Golgi 4.0 %: vacuolar 4.0 %: vesicles of secretory system 4.0 %: e

  14. Dicty_cDB: Contig-U13942-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available sp. AL-2008 voucher USNM:1027763 calmo... 50 0.084 1 ( EU645399 ) Lepidopora micro...420 ) RecName: Full=Putative calmodulin-3; Short=CaM... 49 1e-04 AF020781_1( AF020781 |pid:none) Symbiodinium microadriatic...35185 ;S25133) calmodulin 4 - Arabidopsis thaliana &Z120... 50 5e-05 CP001574_245( CP001574 |pid:none) Micromonas sp. RCC299 chro...217271 |pid:none) Taeniopygia guttata clone 0061P001... 46 3e-04 CP001324_649( CP001324 |pid:none) Micromonas sp. RCC299 chro...66 1e-06 1 ( EU645427 ) Systemapora ornata voucher USNM:1027729 calmoduli... 58 3

  15. Dicty_cDB: Contig-U14946-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available NRRL 5274 ... 81 4e-14 AF020781_1( AF020781 |pid:none) Symbiodinium microadriaticum calmo... 81 4e-14 AL6037... construct calmodulin mut... 93 9e-18 CP001574_245( CP001574 |pid:none) Micromonas sp. RCC299 chro...3e-14 CP001324_649( CP001324 |pid:none) Micromonas sp. RCC299 chromosome... 81 4e...a anserina genomic DNA c... 93 9e-18 (Q95NI4) RecName: Full=Calmodulin; Short=CaM; &...2-326... 79 1e-13 CU633865_49( CU633865 |pid:none) Podospora anserina genomic DNA ch... 79 1e-13 FJ428248_1(

  16. Comparative assessment of the polypeptide profiles from lateral and primary roots of Phaseolus vulgaris L

    Science.gov (United States)

    Westberg, J.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    In Phaseolus vulgaris, primary roots show gravitational sensitivity soon after emerging from the seed. In contrast, lateral roots are agravitropic during early development, and become gravitropic after several cm growth. Primary and lateral root tissues were examined by polyacrylamide gel electrophoresis, coupled with western blotting techniques, to compare proteins which may contribute to the acquisition of gravitational sensitivity. Root tips and zones of cell elongation were compared for each root type, using immunological probes for calmodulin, alpha-actin, alpha-tubulin, and proteins of the plastid envelope. Lateral roots contained qualitatively less calmodulin, and showed a slightly different pattern of actin-related epitope proteins, than did primary root tissues, suggesting that polypeptide differences may contribute to the gravitational sensitivity which these root types express.

  17. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  18. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, A.S.N.; Chengappa, S.; Raghothama, K.G.; Poovaiah, B.W.

    1987-04-01

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (/sup 32/P) PO/sub 4//sup 3 -/ and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone.

  19. Dicty_cDB: Contig-U06073-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 3 2.4 AF084398_1( AF084398 |pid:none) Synthetic construct calmodulin mut... 33 2.4 BC059438_1( BC059438 |pid:none) Danio rerio...m_ : 1.00 44.0 %: cytoplasmic 36.0 %: nuclear 12.0 %: mitochondrial 4.0 %: plasma membrane 4.0 %: peroxisomal >> predictio...hromoso... 32 9.2 AF020781_1( AF020781 |pid:none) Symbiodinium microadriaticum calmo... 32 9.2 CP000586_388(...35 1.1 AJ308296_1( AJ308296 |pid:none) Lycopersicon esculentum mRNA for c... 35 1...1_1( EU959441 |pid:none) Zea mays clone 218171 calmodulin m... 32 5.4 EF652029_1( EF652029 |pid:none) Aspergillus restric

  20. Dicty_cDB: SLI715 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available cctrsfnwwfi**csryvvnwchylyfimwfptilcfis klii*knfnr*l*fprtrmdscl*tcqsfh*klncqrsrstsycqtms**plalw...c... 137 2e-31 (Q8IU85) RecName: Full=Calcium/calmodulin-dependent protein kina...... 137 2e-31 (Q8BW96) RecName: Full=Calcium/calmodulin-dependent protein kina... 137 2e-31 BC041721_1( BC0417...romoso... 148 5e-35 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-CaM-K mRNA f... 142 4e-33 BC074183...SL (Link to library) SLI715 (Link to dictyBase) - - - Contig-U11996-1 SLI715Z (Link to Original

  1. Dicty_cDB: SSD853 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available cfisklii*knfnr*l*fprtrmdscl*t cqsfh*klncqrsr*rsycqtms**pwalw**singfcc*splsfr*kdeei**pkkrws ifk*lnliiiiikkkkk...vis MGC82022 protein, m... 101 7e-21 ( Q63450 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 1...00 1e-20 ( Q14012 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 100 1e-20 PDBF( 1A06 ) MOL_ID...76903 |pid:none) Ciona intestinalis Ci-CaM-K mRNA f... 103 2e-21 BC074183_1( BC074183 |pid:none) Xenopus lae...SS (Link to library) SSD853 (Link to dictyBase) - - - Contig-U11996-1 SSD853Z (Link to Original

  2. Dicty_cDB: SSB277 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ence (All Frames) Frame A: vvnwchylyfimwfptilcfisklii*knfnr*l*fprtrmdscl*tcqsfh*klncqrs rstsycqtms**plalw...0 AK052401_1( AK052401 |pid:none) Mus musculus 13 days embryo lung c... 98 1e-19 (Q8IU85) RecName: Full=Calcium/cal... Synthetic construct Homo sapiens C... 98 1e-19 (Q8BW96) RecName: Full=Calcium/calmodulin-dependent protein ...kina... 98 1e-19 ( Q63450 ) RecName: Full=Calcium/calmodulin-dependent protein kina... 97 3e-19 ( Q14012 ) RecName: Full=Calcium/cal...C82022 protein, m... 100 2e-20 AB076903_1( AB076903 |pid:none) Ciona intestinalis Ci-CaM-K mRNA f... 99 8e-2

  3. The calcium mobilizing tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion

    DEFF Research Database (Denmark)

    Thastrup, Ole; Foder, B; Scharff, O

    1987-01-01

    The ability of the platelet agonists thapsigargin (Tg) and thrombin to elevate the cytoplasmic free calcium level ([Ca2+]i) was examined. Both agonists induced a transient increase of [Ca2+]i with a different time-course, however. Thus, the maximal [Ca2+]i was reached 15 sec and 2 min after...... stimulation with thrombin and Tg, respectively. The thrombin induced rise of [Ca2+]i was reversible, which indicates that active calcium sequestration and/or extrusion is operating. Tg affected [Ca2+]i in a divergent manner, thus, [Ca2+]i was stabilized on a elevated level without initial formation of a...... pronounced peak. The decline in [Ca2+]i observed after thrombin stimulation was not impaired by the calmodulin binding drug trifluoperazine but it was strongly reduced by vanadate, which suggests the active calcium transport systems to be insensitive to calmodulin. We put forward the hypothesis that the...

  4. Association of Protein Phosphatase 1γ1 with Spinophilin Suppresses Phosphatase Activity in a Parkinson Disease Model*

    OpenAIRE

    Brown, Abigail M.; Baucum, Anthony J.; Bass, Martha A.; Roger J Colbran

    2008-01-01

    Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr286 phosphorylation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks...

  5. The second messenger system(s) mediating the secretion of atrial natriuretic peptide (ANP) from the isolated rat heart during rapid cardiac pacing.

    Science.gov (United States)

    Doubell, A F

    1989-01-01

    This study demonstrates that rapid cardiac pacing elevates Atrial Natriuretic Peptide (ANP) levels, independently from atrial stretch. The second messenger system mediating this response was examined. The phosphoinositide system, generally regarded to be important in mediating ANP release, was shown to play only a modulating role during rapid cardiac pacing. The main mediator would appear to be calcium, and a non-calmodulin dependent, calcium mediated system controlling ANP release during rapid cardiac pacing is suggested. PMID:2532285

  6. Electric field-directed cell shape changes, displacement, and cytoskeletal reorganization are calcium dependent

    OpenAIRE

    1988-01-01

    C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field- induced disorganization of the stress fiber pattern, which was reduced when stimulation wa...

  7. Effects of phosphorylation on function of the Rad GTPase.

    Science.gov (United States)

    Moyers, J S; Zhu, J; Kahn, C R

    1998-08-01

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases. PMID:9677319

  8. Search for eukaryotic motility proteins in spirochetes: immunological detection of a tektin-like protein in Spirochaeta halophila.

    Science.gov (United States)

    Barth, A L; Stricker, J A; Margulis, L

    1991-01-01

    The serial endosymbiotic theory (SET) for the spirochete origin of undulipodia (cilia and eukaryotic flagella) predicts that a greater number of axonemal proteins will show homology to spirochete than to other bacterial proteins. To continue testing, the SET proteins associated with eukaryotic motility (tektin, centrin and calmodulin) were sought in Spirochaeta halophila. Western blot immunological detection techniques (for tektin and centrin) and two-dimensional gel analysis (for calmodulin) were used. Tektins are filamentous proteins extending the length of the axoneme in sperm tails and other undulipodia. Whole cell extracts of S. halophila were probed with antibodies made against three sea urchin (Lytechinus pictus) sperm axonemal tektins (tektins A, B, and C). In the spirochetes, one tektin-like protein was detected as a band on Western blots (a C tektin.) An antibody made against Lytechinus pictus sperm tail axonemes, affinity-purified against the C tektin of another sea urchin, Stronglyocentrotus purpuratus, bound to a 30 kDa protein from Spirochaeta halophila. The C tektin epitope was not detected in Escherichia coli. Both the poly- and monoclonal anti-centrin antibodies cross-reacted with multiple proteins in the control alga (Tetraselmis striata) and in the putatively negative control bacterium E. coli. No cross reaction was seen between any anti-centrin antibody and S. halophila. Neither did a two-dimensional gel analysis reveal the presence of calmodulin in these spirochetes or in the two other prokaryotes tested (Spiroplasma citri, Acholeplasma laidlawii). Although neither centrin nor calmodulin were detected, a 30 kDa tektin-like protein apparently is present in these spirochetes. PMID:1863720

  9. Characterization of Tamoxifen as an Antifungal Agent Using the Yeast Schizosaccharomyces Pombe Model Organism

    OpenAIRE

    Zhang, Xibo; Fang, Yue; Jaiseng, Wurentuya; Hu, Lingling; Lu, Yabin; Ma, Yan; Furuyashiki, Tomoyuki

    2015-01-01

    Tamoxifen, a selective estrogen receptor modulator used for managing breast cancer, is known to have antifungal activity. However, its molecular mechanism remains unknown. Using the fission yeast Schizosaccharomyces pombe as a model organism, we have explored the mechanism involved in antifungal action of tamoxifen. Since tamoxifen was shown to inhibit the binding of calmodulin to calcineurin in fungi, we first examined involvement of these molecules and found that overexpression of a catalyt...

  10. Molecular analysis of adenylyl cyclase: Bacillus anthracis edema factor exotoxin

    OpenAIRE

    Mohammed, Hesham Hamada Taha

    2010-01-01

    Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins, i.e. lethal factor, protective antigen and edema factor EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic- resistant strains. Thus, more effective drugs for anthrax treatment are needed. We successfully purified the recombinant full-length EF and EF3(F586A) from E. coli with...

  11. Derivation of neural stem cells from an animal model of psychiatric disease

    OpenAIRE

    Koning, A.; Walton, N M; Shin, R.; Q. Chen; Miyake, S.; Tajinda, K; Gross, A K; Kogan, J H; Heusner, C L; Tamura, K.; Matsumoto, M.

    2013-01-01

    Several psychiatric and neurological diseases are associated with altered hippocampal neurogenesis, suggesting differing neural stem cell (NSC) function may play a critical role in these diseases. To investigate the role of resident NSCs in a murine model of psychiatric disease, we sought to isolate and characterize NSCs from alpha-calcium-/calmodulin-dependent protein kinase II heterozygous knockout (CaMK2α-hKO) mice, a model of schizophrenia/bipolar disorder. These mice display altered neur...

  12. Integration of developmental and environmental signals via a polyadenylation factor in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Man Liu

    Full Text Available The ability to integrate environmental and developmental signals with physiological responses is critical for plant survival. How this integration is done, particularly through posttranscriptional control of gene expression, is poorly understood. Previously, it was found that the 30 kD subunit of Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF30 is a calmodulin-regulated RNA-binding protein. Here we demonstrated that mutant plants (oxt6 deficient in AtCPSF30 possess a novel range of phenotypes--reduced fertility, reduced lateral root formation, and altered sensitivities to oxidative stress and a number of plant hormones (auxin, cytokinin, gibberellic acid, and ACC. While the wild-type AtCPSF30 (C30G was able to restore normal growth and responses, a mutant AtCPSF30 protein incapable of interacting with calmodulin (C30GM could only restore wild-type fertility and responses to oxidative stress and ACC. Thus, the interaction with calmodulin is important for part of AtCPSF30 functions in the plant. Global poly(A site analysis showed that the C30G and C30GM proteins can restore wild-type poly(A site choice to the oxt6 mutant. Genes associated with hormone metabolism and auxin responses are also affected by the oxt6 mutation. Moreover, 19 genes that are linked with calmodulin-dependent CPSF30 functions, were identified through genome-wide expression analysis. These data, in conjunction with previous results from the analysis of the oxt6 mutant, indicate that the polyadenylation factor AtCPSF30 is a regulatory hub where different signaling cues are transduced, presumably via differential mRNA 3' end formation or alternative polyadenylation, into specified phenotypic outcomes. Our results suggest a novel function of a polyadenylation factor in environmental and developmental signal integration.

  13. AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells

    OpenAIRE

    Kim, Nami; Lee, Jung Ok; Lee, Hye Jeong; Lee, Yong Woo; Kim, Hyung Ip; Kim, Su Jin; Park, Sun Hwa; Lee, Chul Su; Ryoo, Sun Woo; Hwang, Geum-Sook; Kim, Hyeon Soo

    2016-01-01

    Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phospho...

  14. Sodium Permeability of a Cloned Small-Conductance Calcium-Activated Potassium Channel

    OpenAIRE

    Shin, Narae; Soh, Heun; Chang, Sunghoe; Kim, Do Han; Park, Chul-Seung

    2005-01-01

    Small-conductance Ca2+-activated potassium channels (SKCa channels) are heteromeric complexes of pore-forming main subunits and constitutively bound calmodulin. SKCa channels in neuronal cells are activated by intracellular Ca2+ that increases during action potentials, and their ionic currents have been considered to underlie neuronal afterhyperpolarization. However, the ion selectivity of neuronal SKCa channels has not been rigorously investigated. In this study, we determined the monovalent...

  15. Effect of maternal manganese blood levels on erythrocyte calcium-pump activity in newborns

    OpenAIRE

    Yazbeck, Chadi; Moreau, Thierry; Sahuquillo, Josiane; Takser, Larissa; Huel, Guy

    2006-01-01

    Manganese (Mn) is widely distributed in the biosphere but occurs in only trace amounts in animal tissues. Although Mn deficiency and toxicity both have pathological consequences, the underlying biochemical lesions have not been well defined. In vitro studies suggest that transport proteins are affected by Mn, lead (Pb), and selenium (Se). Among these transport proteins, the calmodulin-regulated calcium pump (Ca(2+)Mg(2+)ATPase) could be inhibited by Mn. In order to understand Mn biochemical p...

  16. Hair Mercury Negatively Correlates with Calcium Pump Activity in Human Term Newborns and Their Mothers at Delivery

    OpenAIRE

    Huel, Guy; Sahuquillo, Josiane; Debotte, Ginette; Oury, Jean-François; Takser, Larissa

    2007-01-01

    Background Calcium homeostasis is a known target of several environmental toxicants including lead and mercury. Objective Our goal was to determine the relationship between Hg exposure and erythrocyte Ca pump activity in women at delivery and in their newborns. Methods We determined total Hg as well as Pb concentrations in 81 hair and blood samples obtained at delivery. Basal and calmodulin-stimulated Ca pump activity was measured in red blood cells from cord blood and maternal erythrocyte pl...

  17. Molecular Structure and Target Recognition of Neuronal Calcium Sensor Proteins

    OpenAIRE

    James Ames; Mitsuhiko Ikura

    2011-01-01

    Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulates synaptic acti...

  18. Brassica napus Genome Possesses Extraordinary High Number of CAMTA Genes and CAMTA3 Contributes to PAMP Triggered Immunity and Resistance to Sclerotinia sclerotiorum

    OpenAIRE

    Rahman, Hafizur; Xu, You-Ping; Zhang, Xuan-Rui; Cai, Xin-Zhong

    2016-01-01

    Calmodulin-binding transcription activators (CAMTAs) play important roles in various plant biological processes including disease resistance and abiotic stress tolerance. Oilseed rape (Brassica napus L.) is one of the most important oil-producing crops worldwide. To date, compositon of CAMTAs in genomes of Brassica species and role of CAMTAs in resistance to the devastating necrotrophic fungal pathogen Sclerotinia sclerotiorum are still unknown. In this study, 18 CAMTA genes were identified i...

  19. Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells.

    OpenAIRE

    Redmon, J B; Olson, L K; Armstrong, M B; Greene, M J; Robertson, R. P.

    1996-01-01

    FK506 (tacrolimus) is an immunosuppressive drug which interrupts Ca2+-calmodulin-calcineurin signaling pathways in T lymphocytes, thereby blocking antigen activation of T cell early activation genes. Regulation of insulin gene expression in the beta cell may also involve Ca2+-signaling pathways and FK506 has been associated with insulin-requiring diabetes mellitus during clinical use. The purpose of this study was to characterize the effects of FK506 on human insulin gene transcription, insul...

  20. Changes in the levels of CAM kinase II and synapsin I caused by oxidative stress in the rat brain, and its prevention by vitamin E

    OpenAIRE

    Nozomi Kaneai; Koji Fukui; Taisuke Koike; Shiro Urano

    2012-01-01

    To define whether oxidative stress and aging induce abnormal dissociation of neurotransmitter-enclosing synaptic vesicles in rat brain nerve terminals, we assessed the activation of Ca+/calmodulin dependent protein kinase II (CAM kinase II) and changes in the levels of synapsin I, which is a synaptic vesicle-associated protein involved in the modulation of neurotransmitter release. Assessment of young rats subjected to hyperoxia-induced oxidative stress and normal aged ...

  1. CaMKII Activity in the Ventral Tegmental Area Gates Cocaine-Induced Synaptic Plasticity in the Nucleus Accumbens

    OpenAIRE

    Liu, Xiaojie; Liu, Yong; Zhong, Peng; Wilkinson, Brianna; Qi, Jinshun; Olsen, Christopher M; Bayer, K. Ulrich; Liu, Qing-song

    2013-01-01

    Addictive drugs such as cocaine induce synaptic plasticity in discrete regions of the reward circuit. The aim of the present study is to investigate whether cocaine-evoked synaptic plasticity in the ventral tegmental area (VTA) and nucleus accumbens (NAc) is causally linked. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of long-term synaptic plasticity, learning, and drug addiction. We examined whether blocking CaMKII activity in the VTA affected cocaine conditio...

  2. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    downstream of changes in intracellular Ca(2+) and energy turnover. In particular, a signaling cascade involving Ca(2+)-calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work with...... available and new techniques will undoubtedly reveal the functional significance and signaling mechanisms behind changes in skeletal muscle protein turnover during exercise. Key words: Exercise, skeletal muscle, protein metabolism, translation....

  3. The 3′-untranslated region of CaMKIIα is a cis-acting signal for the localization and translation of mRNA in dendrites

    OpenAIRE

    Mayford, Mark; Baranes, Danny; Podsypanina, Katrina; Kandel, Eric R.

    1996-01-01

    Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The α subunit of Ca2+-calmodulin-dependent protein kinase II (CaMKIIα) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subc...

  4. Lessons in Protein Design from Combined Evolution and Conformational Dynamics

    OpenAIRE

    Swarnendu Tripathi; M Neal Waxham; Cheung, Margaret S.; Yin Liu

    2015-01-01

    Protein-protein interactions play important roles in the control of every cellular process. How natural selection has optimized protein design to produce molecules capable of binding to many partner proteins is a fascinating problem but not well understood. Here, we performed a combinatorial analysis of protein sequence evolution and conformational dynamics to study how calmodulin (CaM), which plays essential roles in calcium signaling pathways, has adapted to bind to a large number of partne...

  5. The IQ domain drives nuclear translocation of Nuclear myosin I

    Czech Academy of Sciences Publication Activity Database

    Dzijak, Rastislav; Kahle, Michal; Přidalová, Jarmila; Moško, Tibor; Hozák, Pavel

    St Andrews : Wilhelm Bernhard Workshop, 2007. ---. [Wilhelm Bernhard Workshop on the Cell Nucleus /20./. 27.08.2007-31.08.2007, St. Andrews] R&D Projects: GA MŠk LC545; GA ČR(CZ) GA204/07/1592; GA ČR GD204/05/H023 Institutional research plan: CEZ:AV0Z50520514 Keywords : nucleus * nuclear myosin * calmodulin Subject RIV: EB - Genetics ; Molecular Biology

  6. Pharmacologic study of calcium influx pathways in rabbit aortic smooth muscle

    International Nuclear Information System (INIS)

    Functional characteristics and pharmacologic domains of receptor-operated and potential-sensitive calcium (Ca2+) channels (ROCs and PSCs, respectively) were derived via measurements of 45Ca2+ influx (M/sup Ca/) during activation by the neurotransmitters norepinephrine (NE), histamine (HS), and serotonin (5-HT) and by elevated extracellular potassium (K+) in the individual or combined presence of organic Ca2+ channel antagonists (CAts), calmodulin antagonists (Calm-ants), lanthanum (La3+), and agents that increase intracellular levels of cyclic AMP

  7. Scotopic Visual Signaling in the Mouse Retina Is Modulated by High-Affinity Plasma Membrane Calcium Extrusion

    OpenAIRE

    Duncan, Jacque L.; Yang, Haidong; Doan, Thuy; Silverstein, Robert S.; Murphy, Gabe J.; Nune, George; Liu, Xiaorong; Copenhagen, David; Tempel, Bruce L; Rieke, Fred; KRIŽAJ, DAVID

    2006-01-01

    Transmission of visual signals at the first retinal synapse is associated with changes in calcium concentration in photoreceptors and bipolar cells. We investigated how loss of plasma membrane Ca2+ ATPase isoform 2 (PMCA2), the calcium transporter isoform with the highest affinity for Ca2+/calmodulin, affects transmission of rod- and cone-mediated responses. PMCA2 expression in the neuroblast layer was observed soon after birth; in the adult, PMCA2 was expressed in inner segments and synaptic...

  8. Haloperidol Disrupts Opioid-Antinociceptive Tolerance and Physical Dependence

    OpenAIRE

    Yang, Cheng; Chen, Yan; Tang, Lei; Wang, Zaijie Jim

    2011-01-01

    Previous studies from our laboratory and others have implicated a critical role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in opioid tolerance and dependence. Translational research targeting the CaMKII pathway is challenging, if not impossible, because of a lack of selective inhibitors. We discovered in a preliminary study that haloperidol, a butyrophenone antipsychotic drug, inhibited CaMKII, which led us to hypothesize that haloperidol can attenuate opioid tolerance and depend...

  9. Two new aflatoxin producing species, and an overview of Aspergillus section Flavi

    DEFF Research Database (Denmark)

    Varga, J.; Frisvad, Jens Christian; Samson, R. A.

    2011-01-01

    Aspergillus subgenus Circumdati section Flavi includes species with usually biseriate conidial heads, in shades of yellow-green to brown, and dark sclerotia. Several species assigned to this section are either important mycotoxin producers including aflatoxins, cyclopiazonic acid, ochratoxins and...... kojic acid, or are used in oriental food fermentation processes and as hosts for heterologous gene expression. A polyphasic approach was applied using morphological characters, extrolite data and partial calmodulin, beta-tubulin and ITS sequences to examine the evolutionary relationships within this...

  10. Synaptic Homeostasis and Restructuring across the Sleep-Wake Cycle

    OpenAIRE

    Wilfredo Blanco; Catia M Pereira; Vinicius R Cota; Annie C Souza; César Rennó-Costa; Sharlene Santos; Gabriella Dias; Guerreiro, Ana M. G.; Tort, Adriano B. L.; Adrião D Neto; Sidarta Ribeiro

    2015-01-01

    Sleep is critical for hippocampus-dependent memory consolidation. However, the underlying mechanisms of synaptic plasticity are poorly understood. The central controversy is on whether long-term potentiation (LTP) takes a role during sleep and which would be its specific effect on memory. To address this question, we used immunohistochemistry to measure phosphorylation of Ca2+/calmodulin-dependent protein kinase II (pCaMKIIα) in the rat hippocampus immediately after specific sleep-wake states...

  11. CAMTA 1 regulates drought responses in Arabidopsis thaliana

    OpenAIRE

    Pandey, Neha; Ranjan, Alok; Pant, Poonam; Tripathi, Rajiv K; Ateek, Farha; Pandey, Haushilla P; Patre, Uday V; Sawant, Samir V

    2013-01-01

    Background Transcription factors (TF) play a crucial role in regulating gene expression and are fit to regulate diverse cellular processes by interacting with other proteins. A TF named calmodulin binding transcription activator (CAMTA) was identified in Arabidopsis thaliana (AtCAMTA1-6). To explore the role of CAMTA1 in drought response, the phenotypic differences and gene expression was studied between camta1 and Col-0 under drought condition. Results In camta1, root development was abolish...

  12. Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes*

    OpenAIRE

    Lysechko, Tara L.; Cheung, Samuel M. S.; Ostergaard, Hanne L.

    2010-01-01

    Pyk2 was identified as a Ca2+-dependent kinase, however, the regulation of Pyk2 by Ca2+ in T cells remains controversial. We found that Ca2+ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca2+ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the cal...

  13. Slow motility in hair cells of the frog amphibian papilla: Myosin light chain-mediated shape change

    OpenAIRE

    Farahbakhsh, Nasser A.; Narins, Peter M.

    2008-01-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca2+/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were ...

  14. Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes.

    OpenAIRE

    Luo, C.; Burgeon, E; Carew, J A; McCaffrey, P G; Badalian, T M; Lane, W S; Hogan, P G; Rao, A

    1996-01-01

    Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive a...

  15. Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

    OpenAIRE

    Tzavara, E T; Pouille, Y; Defer, N; Hanoune, J

    1996-01-01

    Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activate...

  16. CANPMR syndrome and chromosome 1p32-p31 deletion syndrome coexist in two related individuals affected by simultaneous haplo-insufficiency of CAMTA1 and NIFA genes

    OpenAIRE

    Coci, Emanuele G.; Koehler, Udo; Liehr, Thomas; Stelzner, Armin; Fink, Christian; Langen, Hendrik; Riedel, Joachim

    2016-01-01

    Background Non-progressive cerebellar ataxia with mental retardation (CANPMR, OMIM 614756) and chromosome 1p32-p31 deletion syndrome (OMIM 613735) are two very rare inherited disorders, which are caused by mono-allelic deficiency (haplo-insufficiency) of calmodulin-binding transcription activator 1 (CAMTA1) and, respectively, nuclear factor 1 A (NFIA) genes. The yet reported patients affected by mono-allelic CAMTA1 dysfunction presented with neonatal hypotonia, delayed and ataxic gait, cerebe...

  17. Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

    OpenAIRE

    Yuanxing Gu; Baozhu Qi; Yingshan Zhou; Xiaowu Jiang; Xian Zhang; Xiaoliang Li; Weihuan Fang

    2016-01-01

    Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kin...

  18. Terbium luminescence-lifetime heterogeneity and protein equilibrium conformational dynamics.

    OpenAIRE

    Austin, R H; Stein, D. L.; Wang, J.

    1987-01-01

    The fluorescence decay of the rare earth terbium when bound to the protein calmodulin changes from a simple exponential decay to a complex nonexponential decay as the temperature is lowered below 200 K. We have fit the observed decay curves by assuming that the terbium emission is a forced electric dipole transition and proteins have a distribution of continuous conformational states. Quantitative fits to the data indicate that the root-mean-square configurational deviation of the atoms surro...

  19. Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

    OpenAIRE

    Fruman, D A; Pai, S Y; Burakoff, S J; Bierer, B E

    1995-01-01

    The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substi...

  20. The select action of hippocampal CAMKII in mediating exercise-enhanced cognitive function

    OpenAIRE

    Vaynman, Shoshanna; Ying, Zhe; Gomez-Pinilla, Fernando

    2006-01-01

    We found that a single week of exercise enhanced cognitive function on the Morris water maze (MWM), such that exercise animals were significantly better than sedentary controls at learning and recalling the location of the platform. In order to elucidate the role that calcium calmodulin protein kinase II (CAMKII) holds in mediating the exercise-induced enhancement in learning and memory, a specific antagonist of CAMKII, KN-62, was used to block CAMKII in the hippocampus during a 1-week volunt...