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Sample records for oxidized calmodulin kinase

  1. Endothelial thrombomodulin induces Ca2+ signals and nitric oxide synthesis through epidermal growth factor receptor kinase and calmodulin kinase II.

    PubMed

    David-Dufilho, Monique; Millanvoye-Van Brussel, Elisabeth; Topal, Gokce; Walch, Laurence; Brunet, Annie; Rendu, Francine

    2005-10-28

    Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

  2. Particulate air pollution induces arrhythmia via oxidative stress and calcium calmodulin kinase II activation

    SciTech Connect

    Kim, Jin-Bae; Kim, Changsoo; Choi, Eunmi; Park, Sanghoon; Park, Hyelim; Pak, Hui-Nam; Lee, Moon-Hyoung; Shin, Dong Chun; Hwang, Ki-Chul; Joung, Boyoung

    2012-02-15

    Ambient particulate matter (PM) can increase the incidence of arrhythmia. However, the arrhythmogenic mechanism of PM is poorly understood. This study investigated the arrhythmogenic mechanism of PM. In Sprague–Dawley rats, QT interval was increased from 115.0 ± 14.0 to 142.1 ± 18.4 ms (p = 0.02) after endotracheal exposure of DEP (200 μg/ml for 30 min, n = 5). Ventricular premature contractions were more frequently observed after DEP exposure (100%) than baseline (20%, p = 0.04). These effects were prevented by pretreatment of N-acetylcysteine (NAC, 5 mmol/L, n = 3). In 12 Langendorff-perfused rat hearts, DEP infusion of 12.5 μg/ml for 20 min prolonged action potential duration (APD) at only left ventricular base increasing apicobasal repolarization gradients. Spontaneous early afterdepolarization (EAD) and ventricular tachycardia (VT) were observed in 8 (67%) and 6 (50%) hearts, respectively, versus no spontaneous triggered activity or VT in any hearts before DEP infusion. DEP-induced APD prolongation, EAD and VT were successfully prevented with NAC (5 mmol/L, n = 5), nifedipine (10 μmol/L, n = 5), and active Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) blockade, KN 93 (1 μmol/L, n = 5), but not by thapsigargin (200 nmol/L) plus ryanodine (10 μmol/L, n = 5) and inactive CaMKII blockade, KN 92 (1 μmol/L, n = 5). In neonatal rat cardiomyocytes, DEP provoked ROS generation in dose dependant manner. DEP (12.5 μg/ml) induced apoptosis, and this effect was prevented by NAC and KN 93. Thus, this study shows that in vivo and vitro exposure of PM induced APD prolongation, EAD and ventricular arrhythmia. These effects might be caused by oxidative stress and CaMKII activation. -- Highlights: ► The ambient PM consistently prolonged repolarization. ► The ambient PM induced triggered activity and ventricular arrhythmia. ► These effects were prevented by antioxidants, I{sub CaL} blockade and CaMKII blockade. ► The ambient PM can induce

  3. The Year in Basic Science: calmodulin kinase cascades.

    PubMed

    Means, Anthony R

    2008-12-01

    This article highlights studies published during the past year that represent significant scientific achievements in the world of calmodulin kinase cascades. Calmodulin is the primary receptor for calcium present in all cells. The binding of its calcium ligand results in a conformational change in calmodulin, which allows the calcium-calmodulin complex to interact with many different targets. In the studies to be summarized in this review, the particular calmodulin cascade involved is shown to be the pathway responsible for important biological responses, including long-term memory formation, dendritic cell survival, hypercapnia, neuronal migration, synapse formation, autophagy, fatty acid oxidation, and energy balance. In some cases, the pathway was previously unknown, and the identification of the calmodulin cascade represents the definition of roles. In other cases, manipulating the cascade has suggested therapeutic approaches to certain diseases, most significantly, type 2 diabetes and obesity.

  4. Nitric oxide-activated calcium/calmodulin-dependent protein kinase regulates the abscisic acid-induced antioxidant defence in maize.

    PubMed

    Ma, Fangfang; Lu, Rui; Liu, Huiying; Shi, Ben; Zhang, Jianhua; Tan, Mingpu; Zhang, Aying; Jiang, Mingyi

    2012-08-01

    Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK.

  5. Nitric oxide-activated calcium/calmodulin-dependent protein kinase regulates the abscisic acid-induced antioxidant defence in maize

    PubMed Central

    Zhang, Aying; Jiang, Mingyi

    2012-01-01

    Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK. PMID:22865912

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

    SciTech Connect

    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.

  7. Cross-Linking Proteins To Show Complex Formation: A Laboratory That Visually Demonstrates Calmodulin Binding to Calmodulin Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2003-01-01

    Presents a laboratory experiment demonstrating the binding of calcium/calmodulin to calmodulin kinase II, which is important in the metabolic and physiological activities of the cell. Uses SDS polyacrylamide gel electrophoresis (PAGE). (YDS)

  8. Cross-Linking Proteins To Show Complex Formation: A Laboratory That Visually Demonstrates Calmodulin Binding to Calmodulin Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2003-01-01

    Presents a laboratory experiment demonstrating the binding of calcium/calmodulin to calmodulin kinase II, which is important in the metabolic and physiological activities of the cell. Uses SDS polyacrylamide gel electrophoresis (PAGE). (YDS)

  9. Possible interaction of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II on reversal of spatial memory impairment induced by morphine.

    PubMed

    Farahmandfar, Maryam; Kadivar, Mehdi; Naghdi, Nasser

    2015-03-15

    The opioid system plays an important role in learning and memory by modulation of different molecules in the brain. The aim of the present study was to investigate the role of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II (CaMKII) on the morphine-induced modulation of spatial memory consolidation in male rats. Spatial memory was assessed in Morris water maze task by a single training session of eight trials followed by a probe trial and visible test 24h later. Our data indicated that post-training administration of L-arginine, a nitric oxide precursor (6 and 9 µg/rat, intra-CA1) significantly decreased amnesia induced by morphine (10 mg/kg) in spatial memory consolidation. A reversal effect of L-arginine on morphine-induced amnesia prevented by KN-93 (N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl) phenyl]-N-[2-hydroxyethyl] methoxybenzenesulfnamide), CaMKII inhibitor, (10 nmol/0.5 µl/site). In addition, post-training injection of L-NAME, (NG-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor (10 and 15 µg/rat) or KN-93 (10 nmol/0.5 µl/site) with lower dose of morphine (2.5 mg/kg), which did not induce amnesia by itself, caused inhibition of memory consolidation. We also showed that co-administration of L-arginine (9 µg/rat) and morphine (10 mg/kg) significantly increased CaMKII activity in the rat hippocampus. On the other hand, administration of L-NAME (10 µg/rat) led to a decrease in the haippocampal activity of CaMKII in morphine-treated (2.5mg/kg) animals. These results indicate that acute single exposure to morphine can modulate consolidation of spatial memory, which may be mediated by a hippocampal nitrergic system and CaMKII activity.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  12. Oxidation of calmodulin alters activation and regulation of CaMKII.

    PubMed

    Robison, A J; Winder, Danny G; Colbran, Roger J; Bartlett, Ryan K

    2007-04-27

    Increases in reactive oxygen species and mis-regulation of calcium homeostasis are associated with various physiological conditions and disease states including aging, ischemia, exposure to drugs of abuse, and neurodegenerative diseases. In aged animals, this is accompanied by a reduction in oxidative repair mechanisms resulting in increased methionine oxidation of the calcium signaling protein calmodulin in the brain. Here, we show that oxidation of calmodulin results in an inability to: (1) activate CaMKII; (2) support Thr(286) autophosphorylation of CaMKII; (3) prevent Thr(305/6) autophosphorylation of CaMKII; (4) support binding of CaMKII to the NR2B subunit of the NMDA receptor; and (5) compete with alpha-actinin for binding to CaMKII. Moreover, oxidized calmodulin does not efficiently bind calcium/calmodulin-dependent protein kinase II (CaMKII) in rat brain lysates or in vitro. These observations contrast from past experiments performed with oxidized calmodulin and the plasma membrane calcium ATPase, where oxidized calmodulin binds to, and partially activates the PMCA. When taken together, these data suggest that oxidative stress may perturb neuronal and cardiac function via a decreased ability of oxidized calmodulin to bind, activate, and regulate the interactions of CaMKII.

  13. Targeting of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Colbran, Roger J

    2004-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types. PMID:14653781

  14. Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species.

    PubMed

    Palomeque, Julieta; Rueda, Omar Velez; Sapia, Luciana; Valverde, Carlos A; Salas, Margarita; Petroff, Martin Vila; Mattiazzi, Alicia

    2009-12-04

    Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. Ang II (1 micromol/L) reduced cat/rat myocytes viability by approximately 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca(2+)/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H(2)O(2), even in the presence of the Ca(2+) chelator BAPTA-AM, in myocytes and in EGTA-Ca(2+)-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca(2+) concentrations, suggesting a new mechanism by which ROS reset the Ca(2+) dependence of CaMKII to extremely low Ca(2+) levels.

  15. Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

    PubMed

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function.

  16. Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium-dependent protein kinases in Ulva compressa exposed to copper excess.

    PubMed

    González, Alberto; Cabrera, M de Los Ángeles; Henríquez, M Josefa; Contreras, Rodrigo A; Morales, Bernardo; Moenne, Alejandra

    2012-03-01

    To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H(2)O(2), ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H(2)O(2) increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H(2)O(2) accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H(2)O(2). In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H(2)O(2), and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein

  17. Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess1[C][W][OA

    PubMed Central

    González, Alberto; Cabrera, M. de los Ángeles; Henríquez, M. Josefa; Contreras, Rodrigo A.; Morales, Bernardo; Moenne, Alejandra

    2012-01-01

    To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H2O2) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H2O2, ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H2O2 increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H2O2 accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H2O2. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H2O2, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases. PMID:22234999

  18. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    SciTech Connect

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

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

    NASA Technical Reports Server (NTRS)

    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.

  20. Calcium-dependent but calmodulin-independent protein kinase from soybean. [Glycine max

    SciTech Connect

    Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

    1987-04-01

    A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (greater than or equal to 2 micromolar). The protein kinase activity was stimulated 100-fold by greater than or equal to 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (less than or equal to 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound /sup 45/Ca/sup 2 +/ in the presence of KCl and MgCl/sub 2/, which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.

  1. Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

    PubMed Central

    Sacks, D B; Fujita-Yamaguchi, Y; Gale, R D; McDonald, J M

    1989-01-01

    It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin

  2. Structure of the smooth muscle myosin light-chain kinase calmodulin-binding domain peptide bound to calmodulin

    SciTech Connect

    Roth, S.M.; Schneider, D.M.; Wand, A.J. Univ. of Illinois, Urbana ); Strobel, L.A. ); VanBerkum, M.F.A.; Means, A.R. )

    1991-10-22

    The interaction between the peptide corresponding to the calmodulin-binding domain of smooth muscle myosin light-chain kinase and (Ca{sup 2+}){sub 4}-calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of {sup 15}N-labeled peptide in conjunction with {sup 15}N-edited and {sup 15}N-correlated {sup 1}H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated calmodulin which is in slow exchange with free peptide. The {sup 1}H and {sup 15}N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin.

  3. Cardiac calmodulin kinase: a potential target for drug design.

    PubMed

    Bányász, T; Szentandrássy, N; Tóth, A; Nánási, P P; Magyar, J; Chen-Izu, Y

    2011-01-01

    Therapeutic strategy for cardiac arrhythmias has undergone a remarkable change during the last decades. Currently implantable cardioverter defibrillator therapy is considered to be the most effective therapeutic method to treat malignant arrhythmias. Some even argue that there is no room for antiarrhythmic drug therapy in the age of implantable cardioverter defibrillators. However, in clinical practice, antiarrhythmic drug therapies are frequently needed, because implantable cardioverter defibrillators are not effective in certain types of arrhythmias (i.e. premature ventricular beats or atrial fibrillation). Furthermore, given the staggering cost of device therapy, it is economically imperative to develop alternative effective treatments. Cardiac ion channels are the target of a number of current treatment strategies, but therapies based on ion channel blockers only resulted in moderate success. Furthermore, these drugs are associated with an increased risk of proarrhythmia, systemic toxicity, and increased defibrillation threshold. In many cases, certain ion channel blockers were found to increase mortality. Other drug classes such as ßblockers, angiotensin-converting enzyme inhibitors, aldosterone antagonists, and statins appear to have proven efficacy for reducing cardiac mortality. These facts forced researchers to shift the focus of their research to molecular targets that act upstream of ion channels. One of these potential targets is calcium/calmodulin-dependent kinase II (CaMKII). Several lines of evidence converge to suggest that CaMKII inhibition may provide an effective treatment strategy for heart diseases. (1) Recent studies have elucidated that CaMKII plays a key role in modulating cardiac function and regulating hypertrophy development. (2) CaMKII activity has been found elevated in the failing hearts from human patients and animal models. (3) Inhibition of CaMKII activity has been shown to mitigate hypertrophy, prevent functional remodeling and

  4. Cardiac Calmodulin Kinase: A Potential Target for Drug Design

    PubMed Central

    Bányász, T.; Szentandrássy, N.; Tóth, A.; Nánási, P.P.; Magyar, J.; Chen-Izu, Y.

    2014-01-01

    Therapeutic strategy for cardiac arrhythmias has undergone a remarkable change during the last decades. Currently implantable cardioverter defibrillator therapy is considered to be the most effective therapeutic method to treat malignant arrhythmias. Some even argue that there is no room for antiarrhythmic drug therapy in the age of implantable cardioverter defibrillators. However, in clinical practice, antiarrhythmic drug therapies are frequently needed, because implantable cardioverter defibrillators are not effective in certain types of arrhythmias (i.e. premature ventricular beats or atrial fibrillation). Furthermore, given the staggering cost of device therapy, it is economically imperative to develop alternative effective treatments. Cardiac ion channels are the target of a number of current treatment strategies, but therapies based on ion channel blockers only resulted in moderate success. Furthermore, these drugs are associated with an increased risk of proarrhythmia, systemic toxicity, and increased defibrillation threshold. In many cases, certain ion channel blockers were found to increase mortality. Other drug classes such as β-blockers, angiotensin-converting enzyme inhibitors, aldosterone antagonists, and statins appear to have proven efficacy for reducing cardiac mortality. These facts forced researchers to shift the focus of their research to molecular targets that act upstream of ion channels. One of these potential targets is calcium/calmodulin-dependent kinase II (CaMKII). Several lines of evidence converge to suggest that CaMKII inhibition may provide an effective treatment strategy for heart diseases. (1) Recent studies have elucidated that CaMKII plays a key role in modulating cardiac function and regulating hypertrophy development. (2) CaMKII activity has been found elevated in the failing hearts from human patients and animal models. (3) Inhibition of CaMKII activity has been shown to mitigate hypertrophy, prevent functional remodeling and

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

    PubMed

    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.

  6. NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms.

    PubMed

    Love, Nick R; Pollak, Nadine; Dölle, Christian; Niere, Marc; Chen, Yaoyao; Oliveri, Paola; Amaya, Enrique; Patel, Sandip; Ziegler, Mathias

    2015-02-03

    Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD(+) kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca(2+)/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin.

    PubMed

    Krasel, C; Dammeier, S; Winstel, R; Brockmann, J; Mischak, H; Lohse, M J

    2001-01-19

    G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC). Whereas PKC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms alpha, gamma, and delta. Two-dimensional peptide mapping of PKCalpha-phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulin-binding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PKC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2.

  11. Identification and characterization of CKLiK, a novel granulocyte Ca(++)/calmodulin-dependent kinase.

    PubMed

    Verploegen, S; Lammers, J W; Koenderman, L; Coffer, P J

    2000-11-01

    Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction-based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin-dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34(+) stem cells. CKLiK kinase activity was dependent on Ca(++) and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinasealpha enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca(++)/calmodulin-dependent PMN- specific kinase that may play a role in Ca(++)-mediated regulation of human granulocyte functions.

  12. Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

    PubMed

    Imai, Seiji; Okayama, Naotsuka; Shimizu, Manabu; Itoh, Makoto

    2003-04-04

    SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

  13. Role of Ca2+/calmodulin-dependent kinases in skeletal muscle plasticity.

    PubMed

    Chin, Eva R

    2005-08-01

    In skeletal muscle, the increase in intracellular Ca(2+) resulting from motor activation plays a key role in both contractile activity-dependent and fiber type-specific gene expression. These motor activation-dependent signals are linked to the amplitude and duration of the Ca(2+) transients that are decoded downstream by Ca(2+)-dependent transcriptional pathways. Evidence is mounting that the Ca(2+)/calmodulin-dependent kinases (CaMKs) such as CaMKII play an important role in regulating oxidative enzyme expression, mitochondrial biogenesis, and expression of fiber type-specific myofibrillar proteins. CaMKIV has been shown to promote mitochondrial biogenesis and a mild fast-to-slow fiber type transition but has recently been shown to not be required for activity-dependent changes in muscle phenotype. CaMKII is known to decode frequency-dependent information and is activated during hypertrophic growth and endurance adaptations and also is upregulated during muscle atrophy. CaMKII has also been shown to remain active in a Ca(2+)-independent manner after acute and prolonged exercise, and, therefore, is implicated as a mechanism for muscle memory. This mechanism can sense altered functional demands and trigger activation of an adaptational response that is dose dependently related to the activation level. This class of enzymes may therefore be the ideal decoders of information encoded by the intensity, frequency, and duty cycle of muscle activation and thus translate level of muscle activation into phenotypic adaptations through regulation of important muscle genes.

  14. Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

    PubMed

    Haga, K; Tsuga, H; Haga, T

    1997-02-11

    Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

  15. 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.

    PubMed

    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.

  16. A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase.

    PubMed

    Win, N H; Ishikawa, T; Saito, N; Kato, M; Yokokura, H; Watanabe, Y; Iida, Y; Hidaka, H

    1996-03-28

    HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide

  17. Tyrosine kinase activity of a Ca{sup 2+}/calmodulin-dependent protein kinase II catalytic fragment

    SciTech Connect

    Sugiyama, Yasunori; Ishida, Atsuhiko; Sueyoshi, Noriyuki; Kameshita, Isamu

    2008-12-12

    A 30-kDa fragment of Ca{sup 2+}/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX{sub 40}-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca{sup 2+}/calmodulin-dependent protein kinase I (CX{sub 40}) were fused to the N-terminal end of 30K-CaMKII. Although CX{sub 40}-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX{sub 40} region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.

  18. Loss of conformational stability in calmodulin upon methionine oxidation.

    PubMed Central

    Gao, J; Yin, D H; Yao, Y; Sun, H; Qin, Z; Schöneich, C; Williams, T D; Squier, T C

    1998-01-01

    We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  1. Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

    PubMed Central

    Banerjee, Chaitali; Khatri, Preeti; Raman, Rajagopal; Bhatia, Himanshi; Datta, Malabika; Mazumder, Shibnath

    2014-01-01

    The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM. PMID:24763432

  2. Impact of methionine oxidation on calmodulin structural dynamics

    SciTech Connect

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin; Moen, Rebecca J.; Olenek, Michael J.; Klein, Jennifer C.; Thomas, David D.

    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

  3. A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum.

    PubMed

    Nakamura, Akio; Hanyuda, Yuki; Okagaki, Tuyoshi; Takagi, Takashi; Kohama, Kazuhiro

    2005-03-25

    A full-length cDNA coding a calmodulin (CaM)-dependent protein kinase gene was cloned from Physarum plasmodia poly(A)-RNA by polymerase chain reaction with the oligonucleotide primers that were designed after the amino acid sequence of highly conserved regions of myosin light-chain kinase. Sequence analysis of the cDNA revealed that this Physarum kinase was a 42,519-Da protein with an ATP-binding domain, Ser/Thr kinase active site signature, and CaM-binding domain. Expression of the cDNA in Escherichia coli demonstrated that the Physarum kinase in the presence of Ca2+ and CaM phosphorylated the recombinant phosphorylatable light chain (PLc) of Physarum myosin II. The peptide analysis after proteolysis of the phosphorylated PLc indicated that Ser 18 was phosphorylated. The site was confirmed by the failure of phosphorylation of PLc, the Ser 18 of which was replaced by Ala. The physiological role of the kinase will be discussed with special reference to the 55-kDa kinase, which had been previously purified from Physarum plasmodia for phosphorylated PLc.

  4. Resveratrol Increases Nitric Oxide Production in the Rat Thick Ascending Limb via Ca2+/Calmodulin

    PubMed Central

    Gonzalez-Vicente, Agustin; Cabral, Pablo D.; Garvin, Jeffrey L.

    2014-01-01

    The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca2+ (Cai) and AMP kinase (AMPK) and NAD-dependent deacetylase sirtuin1 (SIRT1) activities, all of which could activate NO production. We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca2+/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Cai was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick ascending limbs via a Ca2+/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects. PMID:25314136

  5. Calcium/calmodulin-dependent protein kinase II expression in motor neurons: effect of axotomy.

    PubMed

    Lund, L M; McQuarrie, I G

    1997-11-20

    Although Ca2+/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIalpha and IIbeta are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIbeta antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIalpha, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIalpha immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIalpha is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIalpha as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIbeta down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIalpha appears to support axonal regrowth.

  6. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  7. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

    PubMed

    Polya, G M; Chandra, S; Condron, R

    1993-02-01

    A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage.

  11. Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

    PubMed Central

    Polya, G M; Chandra, S; Condron, R

    1993-01-01

    A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage. PMID:8278508

  12. A novel calmodulin-β-PIX interaction and its implication in receptor tyrosine kinase regulation.

    PubMed

    Singh, Vinay K; Munro, Kim; Jia, Zongchao

    2012-09-01

    Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates numerous cellular processes, primarily in response to calcium flux. We have identified and characterized a novel interaction between CaM and β-p21-activated kinase interacting exchange factor (β-PIX), a putative guanine exchange factor implicated in cell signaling, using affinity pull-down assays, co-immunoprecipitation, co-localization and circular dichroism studies. Fluorescence-based titration and isothermal titration calorimetry experiments revealed a Ca(2+)-dependent binding mechanism (K(D)≤10μM). Further, we show that CaM participates in a multi-protein complex involving β-PIX and E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma), which may play a critical role in receptor tyrosine kinase regulation and downstream signaling. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. 2,5-hexanedione (HD) treatment alters calmodulin, Ca{sup 2+}/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

    SciTech Connect

    Wang Qingshan Hou Liyan; Zhang Cuili; Zhao Xiulan; Yu Sufang; Xie, Ke-Qin

    2008-10-01

    Calcium-dependent mechanisms, particularly those mediated by Ca{sup 2+}/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 Ca{sup 2+} 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.

  14. A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin-dependent kinase II holoenzyme

    PubMed Central

    Chao, Luke H.; Stratton, Margaret M.; Lee, Il-Hyung; Rosenberg, Oren S.; Levitz, Joshua; Mandell, Daniel J.; Kortemme, Tanja; Groves, Jay T.; Schulman, Howard; Kuriyan, John

    2011-01-01

    Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains. PMID:21884935

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

    NASA Technical Reports Server (NTRS)

    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.

  16. Calmodulin is a subunit of nitric oxide synthase from macrophages

    PubMed Central

    1992-01-01

    A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist- induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized. PMID:1380065

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  19. Cytokinesis is not controlled by calmodulin or myosin light chain kinase in the Caenorhabditis elegans early embryo

    PubMed Central

    Batchelder, Ellen L.; Thomas–Virnig, Christina L.; Hardin, Jeffery D.; White, John G.

    2007-01-01

    Furrow ingression in animal cell cytokinesis is controlled by phosphorylation of myosin II regulatory light chain (mRLC). In C. elegans embryos, Rho-dependent Kinase (RhoK) is involved in, but not absolutely required for, this phosphorylation. The calmodulin effector Myosin Light Chain Kinase (MLCK) can also phosphorylate mRLC and is widely regarded as a candidate for redundant function with RhoK. However, our results show that RNAi against C. elegans calmodulin and candidate MLCKs had no effect on cytokinesis in wild type or RhoK mutant embryos, ruling out the calmodulin/MLCK pathway as the missing regulator of cytokinesis in the C. elegans early embryo. PMID:17716666

  20. Cytokinesis is not controlled by calmodulin or myosin light chain kinase in the Caenorhabditis elegans early embryo.

    PubMed

    Batchelder, Ellen L; Thomas-Virnig, Christina L; Hardin, Jeffery D; White, John G

    2007-09-04

    Furrow ingression in animal cell cytokinesis is controlled by phosphorylation of myosin II regulatory light chain (mRLC). In Caenorhabditis elegans embryos, Rho-dependent Kinase (RhoK) is involved in, but not absolutely required for, this phosphorylation. The calmodulin effector myosin light chain kinase (MLCK) can also phosphorylate mRLC and is widely regarded as a candidate for redundant function with RhoK. However, our results show that RNA mediated interference against C. elegans calmodulin and candidate MLCKs had no effect on cytokinesis in wild-type or RhoK mutant embryos, ruling out the calmodulin/MLCK pathway as the missing regulator of cytokinesis in the C. elegans early embryo.

  1. Increased calcium/calmodulin-dependent protein kinase II activity by morphine-sensitization in rat hippocampus.

    PubMed

    Kadivar, Mehdi; Farahmandfar, Maryam; Ranjbar, Faezeh Esmaeli; Zarrindast, Mohammad-Reza

    2014-07-01

    Repeated exposure to drugs of abuse, such as morphine, elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect long-lasting changes in some of the important molecules involved in memory processing such as calcium/calmodulin-dependent protein kinase II (CaMKII). In the present study, we investigated the effect of morphine sensitization on mRNA expression of α and β isoforms and activity of CaMKII in the hippocampus of male rats. Animals were treated for 3 days with saline or morphine (20mg/kg) and following a washout period of 5 days, a challenge dose of morphine (5mg/kg) were administered. The results indicate that morphine administration in pre-treated animals produces behavioral sensitization, as determined by significant increase in locomotion and oral stereotypy behavior. In addition, repeated morphine treatment increased mRNA expression of both α and β isoforms of CaMKII in the hippocampus. The present study also showed that induction of morphine sensitization significantly increased both Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent activities of CaMK II in the rat hippocampus. However, acute administration of morphine (5mg/kg) did not alter either α and β CaMKII mRNA expression or CaMKII activity in the hippocampus. The stimulation effects of morphine sensitization on mRNA expression and activity of CaMKII were completely abolished by administration of naloxone, 30min prior to s.c. injections of morphine (20mg/kg/day×3 days). Our data demonstrated that induction of morphine sensitization could effectively modulate the activity and the mRNA expression of CaMKII in the hippocampus and this effect of morphine was exerted by the activation of opioid receptors.

  2. A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.

    PubMed Central

    Charpenteau, Martine; Jaworski, Krzysztof; Ramirez, Bertha C; Tretyn, Andrzej; Ranjeva, Raoul; Ranty, Benoît

    2004-01-01

    Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways. PMID:14720124

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

    EPA Science Inventory

    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...

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

    EPA Science Inventory

    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...

  5. Crystal Structure of the Ca2+/Calmodulin-dependent Protein Kinase Kinase in Complex with the Inhibitor STO-609*

    PubMed Central

    Kukimoto-Niino, Mutsuko; Yoshikawa, Seiko; Takagi, Tetsuo; Ohsawa, Noboru; Tomabechi, Yuri; Terada, Takaho; Shirouzu, Mikako; Suzuki, Atsushi; Lee, Suni; Yamauchi, Toshimasa; Okada-Iwabu, Miki; Iwabu, Masato; Kadowaki, Takashi; Minokoshi, Yasuhiko; Yokoyama, Shigeyuki

    2011-01-01

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca2+ elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro274, which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases. PMID:21504895

  6. Impact of Methionine Oxidation on Calmodulin Structural Dynamics

    PubMed Central

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin; Moen, Rebecca J.; Olenek, Michael J.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    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 population ranging from 1.5 to 3 nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM’s functional interaction with its target proteins by perturbing this Ca-dependent structural shift. PMID:25478640

  7. A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants.

    PubMed

    Yang, Tianbao; Chaudhuri, Shubho; Yang, Lihua; Du, Liqun; Poovaiah, B W

    2010-03-05

    Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369-390 with a K(d) of 25 nm, and the other located at residues 28-112 with a K(d) of 160 nm. Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a, and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling.

  8. Fast Photochemical Oxidation of Proteins (FPOP) for Comparing Structures of Protein/Ligand Complexes: The Calmodulin-peptide Model System

    PubMed Central

    Zhang, Hao; Gau, Brian C.; Jones, Lisa M.; Vidavsky, Ilan; Gross, Michael L.

    2010-01-01

    Fast Photochemical Oxidation of Proteins (FPOP) is a mass-spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive •OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one half the common amino acids on this time scale. Owing to the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex should provide structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows from a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not,. PMID:21142124

  9. Inactivation of Smad-Transforming Growth Factor β Signaling by Ca2+-Calmodulin-Dependent Protein Kinase II

    PubMed Central

    Wicks, Stephen J.; Lui, Stephen; Abdel-Wahab, Nadia; Mason, Roger M.; Chantry, Andrew

    2000-01-01

    Members of the transforming growth factor β (TGF-β) family transduce signals through Smad proteins. Smad signaling can be regulated by the Ras/Erk/mitogen-activated protein pathway in response to receptor tyrosine kinase activation and the gamma interferon pathway and also by the functional interaction of Smad2 with Ca2+-calmodulin. Here we report that Smad–TGF-β-dependent transcriptional responses are prevented by expression of a constitutively activated Ca2+-calmodulin-dependent protein kinase II (Cam kinase II). Smad2 is a target substrate for Cam kinase II in vitro at serine-110, -240, and -260. Cam kinase II induces in vivo phosphorylation of Smad2 and Smad4 and, to a lesser extent, Smad3. A phosphopeptide antiserum raised against Smad2 phosphoserine-240 reacted with Smad2 in vivo when coexpressed with Cam kinase II and by activation of the platelet-derived growth factor receptor, the epidermal growth factor receptor, HER2 (c-erbB2), and the TGF-β receptor. Furthermore, Cam kinase II blocked nuclear accumulation of a Smad2 and induced Smad2-Smad4 hetero-oligomerization independently of TGF-β receptor activation, while preventing TGF-β-dependent Smad2-Smad3 interactions. These findings provide a novel cross-talk mechanism by which Ca2+-dependent kinases activated downstream of multiple growth factor receptors antagonize cell responses to TGF-β. PMID:11027280

  10. Ca(2+)/Calmodulin-Dependent Protein Kinase II in Vascular Smooth Muscle.

    PubMed

    Saddouk, F Z; Ginnan, R; Singer, H A

    2017-01-01

    Ca(2+)-dependent signaling pathways are central regulators of differentiated vascular smooth muscle (VSM) contractile function. In addition, Ca(2+) signals regulate VSM gene transcription, proliferation, and migration of dedifferentiated or "synthetic" phenotype VSM cells. Synthetic phenotype VSM growth and hyperplasia are hallmarks of pervasive vascular diseases including hypertension, atherosclerosis, postangioplasty/in-stent restenosis, and vein graft failure. The serine/threonine protein kinase Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous mediator of intracellular Ca(2+) signals. Its multifunctional nature, structural complexity, diversity of isoforms, and splice variants all characterize this protein kinase and make study of its activity and function challenging. The kinase has unique autoregulatory mechanisms, and emerging studies suggest that it can function to integrate Ca(2+) and reactive oxygen/nitrogen species signaling. Differentiated VSM expresses primarily CaMKIIγ and -δ isoforms. CaMKIIγ isoform expression correlates closely with the differentiated phenotype, and some studies link its function to regulation of contractile activity and Ca(2+) homeostasis. Conversely, synthetic phenotype VSM cells primarily express CaMKIIδ and substantial evidence links it to regulation of gene transcription, proliferation, and migration of VSM in vitro, and vascular hypertrophic and hyperplastic remodeling in vivo. CaMKIIδ and -γ isoforms have opposing functions at the level of cell cycle regulation, proliferation, and VSM hyperplasia in vivo. Isoform switching following vascular injury is a key step in promoting vascular remodeling. Recent availability of genetically engineered mice with smooth muscle deletion of specific isoforms and transgenics expressing an endogenous inhibitor protein (CAMK2N) has enabled a better understanding of CaMKII function in VSM and should facilitate future studies. © 2017 Elsevier Inc. All rights reserved.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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. Bcl10 is phosphorylated on Ser138 by Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    Ishiguro, Kazuhiro; Ando, Takafumi; Goto, Hidemi; Xavier, Ramnik

    2007-03-01

    Ordered assembly of scaffold proteins Carma1-Bcl10-Malt1 determines NF-kappaB activation following T cell receptor (TCR) engagement. Carma1-Bcl10 interaction and the signaling pathway are controlled by Carma1 phosphorylation, which are induced by PKCtheta and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition to Carma1 phosphorylation, previous studies have demonstrated that Bcl10 is phosphorylated in the C-terminal Ser/Thr rich region following TCR engagement. However the kinases that phosphorylate Bcl10 are incompletely understood. Here we show that CaMKII phosphorylates Bcl10 on Ser138. Furthermore, a CaMKII inhibitor, KN93, and CaMKII siRNA substantially reduce Bcl10 phosphorylation induced by phorbol myristate acetate/ionomycin. S138A mutation prolongs Bcl10-induced NF-kappaB activation, suggesting that Bcl10 phosphorylation is involved in attenuation of NF-kappaB activation. These findings suggest that CaMKII modulates NF-kappaB activation via phosphorylating Bcl10 as well as Carma1.

  14. [Gene expression and activity regulation of two calmodulin binding protein kinases in tobacco seedling].

    PubMed

    Hua, Wei; Li, Rong-Jun; Liang, Shu-Ping; Lu, Ying-Tang

    2005-06-01

    Two different calmodulin-binding protein kinase cDNAs (NtCBK1/2) have been isolated from tobacco. To understand the CBK protein activity regulation, we compared the activity regulation of NtCBK1 and NtCBK2 by pH, Mg(2+) concentration and Na(+) concentration. We found the autophosphorylation of NtCBK1/2 reached the maximum in pH 7.5 and 8 respectively; Mg(2+) and Na(+) shown different effects on the activity of NtCBKs, high and low Mg(2+) concentrations both inhibited the activity of NtCBKs, but Na+ had little effect on the kinase activity. In addition, to obtain further insight about the physiological roles of individual NtCBKs, we detected the expression profiles of CBKs. The results revealed different patterns of expression of NtCBK1 and NtCBK2. Both are largely expressed in leaf and flower; but in stem and root, NtCBK1 gene had stronger expression than NtCBK2. NtCBK2 expression was induced by GA treatment, while NtCBK1 expression remained unchanged under GA treatment. Expression of both NtCBK1 and NtCBK2 increased in response to salt stress, the former to a greater extent, and both expressions did not change under high/low temperature, drought, NAA and ABA treatments.

  15. 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

    NASA Technical Reports Server (NTRS)

    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.

  16. Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

    PubMed

    Maubach, Gunter; Sokolova, Olga; Wolfien, Markus; Rothkötter, Hermann-Josef; Naumann, Michael

    2013-09-15

    Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

  17. Ca2+/Calmodulin-Dependent Kinase Kinase α Is Expressed by Monocytic Cells and Regulates the Activation Profile

    PubMed Central

    Guest, Christopher B.; Deszo, Eric L.; Hartman, Matthew E.; York, Jason M.; Kelley, Keith W.; Freund, Gregory G.

    2008-01-01

    Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca2+/calmodulin-dependent kinase kinase α (CaMKKα) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKα by mass spectrometry and Western analysis. The function of CaMKKα in monocyte activation was examined using the CaMKKα inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKα, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKα to the nucleus. Finally, to further examine monocyte activation profiles, TNFα and IL-10 secretion were studied. CaMKKα inhibition attenuated PMA-dependent IL-10 production and enhanced TNFα production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKα in the differentiation of monocytic cells. PMID:18270593

  18. GsCBRLK, a calcium/calmodulin-binding receptor-like kinase, is a positive regulator of plant tolerance to salt and ABA stress.

    PubMed

    Yang, Liang; Ji, Wei; Zhu, Yanming; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Guo, Dianjing

    2010-05-01

    Calcium/calmodulin-dependent kinases play vital roles in protein phosphorylation in eukaryotes, yet little is known about the phosphorylation process of calcium/calmodulin-dependent protein kinase and its role in stress signal transduction in plants. A novel plant-specific calcium-dependent calmodulin-binding receptor-like kinase (GsCBRLK) has been isolated from Glycine soja. A subcellular localization study using GFP fusion protein indicated that GsCBRLK is localized in the plasma membrane. Binding assays demonstrated that calmodulin binds to GsCBRLK with an affinity of 25.9 nM in a calcium-dependent manner and the binding motif lies between amino acids 147 to169 within subdomain II of the kinase domain. GsCBRLK undergoes autophosphorylation and Myelin Basis Protein phosphorylation in the presence of calcium. It was also found that calcium/calmodulin positively regulates GsCBRLK kinase activity through direct interaction between the calmodulin-binding domain and calmodulin. So, it is likely that GsCBRLK responds to an environmental stimulus in two ways: by increasing the protein expression level and by regulating its kinase activity through the calcium/calmodulin complex. Furthermore, cold, salinity, drought, and ABA stress induce GsCBRLK gene transcripts. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA and increased the expression pattern of a number of stress gene markers in response to ABA and high salt. These results identify GsCBRLK as a molecular link between the stress- and ABA-induced calcium/calmodulin signal and gene expression in plant cells.

  19. Ca+2/Calmodulin-Dependent Protein Kinase Mediates Glucose Toxicity-Induced Cardiomyocyte Contractile Dysfunction

    PubMed Central

    Zhang, Rong-Huai; Guo, Haitao; Kandadi, Machender R.; Wang, Xiao-Ming; Ren, Jun

    2012-01-01

    (1) Hyperglycemia leads to cytotoxicity in the heart. Although several theories are postulated for glucose toxicity-induced cardiomyocyte dysfunction, the precise mechanism still remains unclear. (2) This study was designed to evaluate the impact of elevated extracellular Ca2+ on glucose toxicity-induced cardiac contractile and intracellular Ca2+ anomalies as well as the mechanism(s) involved with a focus on Ca2+/calmodulin (CaM)-dependent kinase. Isolated adult rat cardiomyocytes were maintained in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) media for 6-12 hours. Contractile indices were measured including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR90). (3) Cardiomyocytes maintained with HG displayed abnormal mechanical function including reduced PS, ±dL/dt, and prolonged TPS, TR90 and intracellular Ca2+ clearance. Expression of intracellular Ca2+ regulatory proteins including SERCA2a, phospholamban and Na+-Ca2+ exchanger were unaffected whereas SERCA activity was inhibited by HG. Interestingly, the HG-induced mechanical anomalies were abolished by elevated extracellular Ca2+ (from 1.0 to 2.7 mM). Interestingly, the high extracellular Ca2+-induced beneficial effect against HG was abolished by the CaM kinase inhibitor KN93. (4) These data suggest that elevated extracellular Ca2+ protects against glucose toxicity-induced cardiomyocyte contractile defects through a mechanism associated with CaM kinase. PMID:22745633

  20. A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation.

    PubMed

    Franco-Echevarría, Elsa; Baños-Sanz, Jose I; Monterroso, Begoña; Round, Adam; Sanz-Aparicio, Julia; González, Beatriz

    2014-11-01

    IP3-3K [Ins(1,4,5)P3 3-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with Ins(1,3,4,5)P4 regulates Ca2+ homoeostasis. In addition, Ins(1,3,4,5)P4 is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.

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

    PubMed Central

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

    2016-01-01

    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 Ca2+/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 Ca2+/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 Ca2+/CaM, cMLCK has constitutive activity that is stimulated by Ca2+/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

  2. Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28)

    USDA-ARS?s Scientific Manuscript database

    Plant calcium (Ca2+) dependent protein kinases (CPKs) are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a Calmodulin-like domain (CLD) via an autoinhibitory junction (J) and represent the primary Ca2+-dependent protein kinase activities in plant systems. While regulation...

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

    SciTech Connect

    Schallreuter, K.U. . E-mail: k.schallreuter@bradford.ac.uk; Gibbons, N.C.J.; Zothner, C.; Abou Elloof, M.M.; Wood, J.M.

    2007-08-17

    Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10{sup -3} M H{sub 2}O{sub 2}. 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{sup -3}M H{sub 2}O{sub 2} oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H{sub 2}O{sub 2} utilising {sup 45}calcium. 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 H{sub 2}O{sub 2}-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo.

  4. Calcium/calmodulin kinase II activity of hippocampus in kainate-induced epilepsy.

    PubMed Central

    Lee, M. C.; Ban, S. S.; Woo, Y. J.; Kim, S. U.

    2001-01-01

    This study investigated calcium/calmodulin kinase II (CaMKII) activity related to long-standing neuronal injury of the hippocampus in kainate (KA)-induced experimental temporal lobe epilepsy. Epileptic seizure was induced by injection of KA (1 microg/microL) dissolved in phosphate buffer (0.1 M, pH 7.4) into the left amygdala. Clinical seizures, histopathologic changes and CaMKII activity of the hippocampus were evaluated. Characteristic early limbic and late seizures were developed. Hippocampal CaMKII activity increased significantly 4 and 8 weeks after intra-amygdaloid injection of KA, when late seizures developed. The histopathologic changes of the hippocampus included swelling of neuronal cytoplasm with nuclear pyknosis and loss of neurons in CA3 during this period. The increased activity of CaMKII may correlate with appearance of distant damage in the hippocampus. The above results indicate that intra-amygdaloid injection of KA produces excitatory signals for ipsilateral CA3 neurons in the hippocampus and that subsequently increased levels of CaMKII in postsynaptic neurons induce neuronal injury via phosphorylation of N-methyl-D-aspartate type glutamate receptor. PMID:11641537

  5. Hunting increases phosphorylation of calcium/calmodulin-dependent protein kinase type II in adult barn owls.

    PubMed

    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 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.

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

    PubMed Central

    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 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. PMID:25789177

  7. Calmodulin-dependent kinase II regulates osteoblast differentiation through regulation of Osterix.

    PubMed

    Choi, You Hee; Choi, Jun-Ha; Oh, Jae-Wook; Lee, Kwang-Youl

    2013-03-08

    Osterix (Osx), a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. However, the precise molecular signaling mechanisms between Osterix and CaMKII are not known. In this study, we focused on the relationship between Osterix and CaMKII during osteoblast differentiation. We examined the role of the CaMKII pathway in the regulation of protein levels and its transcriptional activity on Osterix. We showed that CaMKII interacts with Osterix by increasing the protein levels and enhancing the transcriptional activity of Osterix. Conversely, CaMKII inhibitor KN-93 decreases the protein levels and increases the stability of Osterix. The siRNA-mediated knockdown of CaMKII decreased the protein levels and transcriptional activity of Osterix. These results suggest that Osterix is a novel target of CaMKII and the activity of Osterix can be modulated by a novel mechanism involving CaMKII during osteoblast differentiation.

  8. Ca2+/calmodulin-dependent protein kinase II function in vascular remodelling.

    PubMed

    Singer, Harold A

    2012-03-15

    Vascular smooth muscle (VSM) undergoes a phenotypic switch in response to injury, a process that contributes to pathophysiological vascular wall remodelling. VSM phenotype switching is a consequence of changes in gene expression, including an array of ion channels and pumps affecting spatiotemporal features of intracellular Ca(2+) signals. Ca(2+) signalling promotes vascular wall remodelling by regulating cell proliferation, motility, and/or VSM gene transcription, although the mechanisms are not clear. In this review, the functions of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in VSM phenotype switching and synthetic phenotype function are considered. CaMKII isozymes have complex structural and autoregulatory properties. Vascular injury in vivo results in rapid changes in CaMKII isoform expression with reduced expression of CaMKIIγ and upregulation of CaMKIIδ in medial wall VSM. SiRNA-mediated suppression of CaMKIIδ or gene deletion attenuates VSM proliferation and consequent neointimal formation. In vitro studies support functions for CaMKII in the regulation of cell proliferation, motility and gene expression via phosphorylation of CREB1 and HDACIIa/MEF2 complexes. These studies support the concept, and provide potential mechanisms, whereby Ca(2+) signalling through CaMKIIδ promotes VSM phenotype transitions and vascular remodelling.

  9. Ca2+/calmodulin-dependent protein kinase II function in vascular remodelling

    PubMed Central

    Singer, Harold A

    2012-01-01

    Vascular smooth muscle (VSM) undergoes a phenotypic switch in response to injury, a process that contributes to pathophysiological vascular wall remodelling. VSM phenotype switching is a consequence of changes in gene expression, including an array of ion channels and pumps affecting spatiotemporal features of intracellular Ca2+ signals. Ca2+ signalling promotes vascular wall remodelling by regulating cell proliferation, motility, and/or VSM gene transcription, although the mechanisms are not clear. In this review, the functions of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in VSM phenotype switching and synthetic phenotype function are considered. CaMKII isozymes have complex structural and autoregulatory properties. Vascular injury in vivo results in rapid changes in CaMKII isoform expression with reduced expression of CaMKIIγ and upregulation of CaMKIIδ in medial wall VSM. SiRNA-mediated suppression of CaMKIIδ or gene deletion attenuates VSM proliferation and consequent neointimal formation. In vitro studies support functions for CaMKII in the regulation of cell proliferation, motility and gene expression via phosphorylation of CREB1 and HDACIIa/MEF2 complexes. These studies support the concept, and provide potential mechanisms, whereby Ca2+ signalling through CaMKIIδ promotes VSM phenotype transitions and vascular remodelling. PMID:22124148

  10. Regulation of gastrointestinal motility by Ca2+/calmodulin-stimulated protein kinase II.

    PubMed

    Perrino, Brian A

    2011-06-15

    Gastrointestinal (GI) motility ultimately depends upon the contractile activity of the smooth muscle cells of the tunica muscularis. Integrated functioning of multiple tissues and cell types, including enteric neurons and interstitial cells of Cajal (ICC) is necessary to generate coordinated patterns of motor activity that control the movement of material through the digestive tract. The neurogenic mechanisms that govern GI motility patterns are superimposed upon intrinsic myogenic mechanisms regulating smooth muscle cell excitability. Several mechanisms regulate smooth muscle cell responses to neurogenic inputs, including the multifunctional Ca(2+)/calmodulin-stimulated protein kinase II (CaMKII). CaMKII can be activated by Ca(2+) transients from both extracellular and intracellular sources. Prolonging the activities of Ca(2+)-sensitive K(+) channels in the plasma membrane of GI smooth muscle cells is an important regulatory mechanism carried out by CaMKII. Phospholamban (PLN) phosphorylation by CaMKII activates the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), increasing both the rate of Ca(2+) clearance from the myoplasm and the frequency of localized Ca(2+) release events from intracellular stores. Overall, CaMKII appears to moderate GI smooth muscle cell excitability. Finally, transcription factor activities may be facilitated by the neutralization of HDAC4 by CaMKII phosphorylation, which may contribute to the phenotypic plasticity of GI smooth muscle cells.

  11. Calmodulin activation of Aurora-A kinase (AURKA) is required during ciliary disassembly and in mitosis.

    PubMed

    Plotnikova, Olga V; Nikonova, Anna S; Loskutov, Yuri V; Kozyulina, Polina Y; Pugacheva, Elena N; Golemis, Erica A

    2012-07-01

    The centrosomal Aurora-A kinase (AURKA) regulates mitotic progression, and overexpression and hyperactivation of AURKA commonly promotes genomic instability in many tumors. Although most studies of AURKA focus on its role in mitosis, some recent work identified unexpected nonmitotic activities of AURKA. Among these, a role for basal body-localized AURKA in regulating ciliary disassembly in interphase cells has highlighted a role in regulating cellular responsiveness to growth factors and mechanical cues. The mechanism of AURKA activation involves interactions with multiple partner proteins and is not well understood, particularly in interphase cells. We show here that AURKA activation at the basal body in ciliary disassembly requires interactions with Ca(2+) and calmodulin (CaM) and that Ca(2+)/CaM are important mediators of the ciliary disassembly process. We also show that Ca(2+)/CaM binding is required for AURKA activation in mitosis and that inhibition of CaM activity reduces interaction between AURKA and its activator, NEDD9. Finally, mutated derivatives of AURKA impaired for CaM binding and/or CaM-dependent activation cause defects in mitotic progression, cytokinesis, and ciliary resorption. These results define Ca(2+)/CaM as important regulators of AURKA activation in mitotic and nonmitotic signaling.

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

    PubMed Central

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

    2010-01-01

    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 cell hypertrophy in vitro and in vivo. Specifically, systemic CaMKII inhibition with KN-93 prevented ANG II-mediated hypertension and medial hypertrophy in vivo. Adenoviral transduction with the CaMKII peptide inhibitor CaMKIIN abrogated ANG II-induced VSM hypertrophy in vitro, which was augmented by overexpression of CaMKII-δ2. Finally, we identify the downstream signaling components critical for ANG II- and CaMKII-mediated VSM hypertrophy. Specifically, we demonstrate that CaMKII induces VSM hypertrophy by regulating histone deacetylase 4 (HDAC4) activity, thereby stimulating activity of the hypertrophic transcription factor MEF2. MEF2 transcription is activated by ANG II in vivo and abrogated by the CaMKII inhibitor KN-93. Together, our studies identify a complete pathway for ANG II-triggered arterial VSM hypertrophy and identify new potential therapeutic targets for chronic human hypertension. PMID:20023119

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

    PubMed

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

    2010-02-01

    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 cell hypertrophy in vitro and in vivo. Specifically, systemic CaMKII inhibition with KN-93 prevented ANG II-mediated hypertension and medial hypertrophy in vivo. Adenoviral transduction with the CaMKII peptide inhibitor CaMKIIN abrogated ANG II-induced VSM hypertrophy in vitro, which was augmented by overexpression of CaMKII-delta2. Finally, we identify the downstream signaling components critical for ANG II- and CaMKII-mediated VSM hypertrophy. Specifically, we demonstrate that CaMKII induces VSM hypertrophy by regulating histone deacetylase 4 (HDAC4) activity, thereby stimulating activity of the hypertrophic transcription factor MEF2. MEF2 transcription is activated by ANG II in vivo and abrogated by the CaMKII inhibitor KN-93. Together, our studies identify a complete pathway for ANG II-triggered arterial VSM hypertrophy and identify new potential therapeutic targets for chronic human hypertension.

  14. Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation.

    PubMed

    Kanatsu, Yoshinori; Chen, Nai Hong; Mitoma, Junya; Nakagawa, Tetsuto; Hirabayashi, Yoshio; Higashi, Hideyoshi

    2012-07-01

    Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.

  15. Dissecting cooperative calmodulin binding to CaM kinase II: a detailed stochastic model.

    PubMed

    Byrne, Michael J; Putkey, John A; Waxham, M Neal; Kubota, Yoshihisa

    2009-12-01

    Calmodulin (CaM) is a major Ca(2+) binding protein involved in two opposing processes of synaptic plasticity of CA1 pyramidal neurons: long-term potentiation (LTP) and depression (LTD). The N- and C-terminal lobes of CaM bind to its target separately but cooperatively and introduce complex dynamics that cannot be well understood by experimental measurement. Using a detailed stochastic model constructed upon experimental data, we have studied the interaction between CaM and Ca(2+)-CaM-dependent protein kinase II (CaMKII), a key enzyme underlying LTP. The model suggests that the accelerated binding of one lobe of CaM to CaMKII, when the opposing lobe is already bound to CaMKII, is a critical determinant of the cooperative interaction between Ca(2+), CaM, and CaMKII. The model indicates that the target-bound Ca(2+) free N-lobe has an extended lifetime and may regulate the Ca(2+) response of CaMKII during LTP induction. The model also reveals multiple kinetic pathways which have not been previously predicted for CaM-dissociation from CaMKII.

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

    PubMed Central

    Chen, Zhang-Fan; Wang, Hao; Matsumura, Kiyotaka; Qian, Pei-Yuan

    2012-01-01

    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 Ca2+/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. PMID:22348072

  17. Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells.

    PubMed

    Borodinsky, Laura N; Coso, Omar A; Fiszman, Mónica L

    2002-03-01

    In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

  18. Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II

    PubMed Central

    Bhattacharyya, Moitrayee; Stratton, Margaret M; Going, Catherine C; McSpadden, Ethan D; Huang, Yongjian; Susa, Anna C; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L; Barros, Tiago; Schulman, Howard; Williams, Evan R; Kuriyan, John

    2016-01-01

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. DOI: http://dx.doi.org/10.7554/eLife.13405.001 PMID:26949248

  19. Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

    DOE PAGES

    Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.; ...

    2016-03-07

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts themore » hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.« less

  20. Molecular mechanism of activation-triggered subunit exchange in Ca 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Bhattacharyya, Moitrayee; Stratton, Margaret M.; Going, Catherine C.; McSpadden, Ethan D.; Huang, Yongjian; Susa, Anna C.; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L.; Barros, Tiago; Schulman, Howard; Williams, Evan R.; Kuriyan, John

    2016-03-07

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.

  1. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

    PubMed

    Cabell, L; Audesirk, G

    1993-06-01

    A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every

  2. Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION.

    PubMed

    Tavares, Clint D J; Giles, David H; Stancu, Gabriel; Chitjian, Catrina A; Ferguson, Scarlett B; Wellmann, Rebecca M; Kaoud, Tamer S; Ghose, Ranajeet; Dalby, Kevin N

    2017-02-03

    Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca(2+) ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca(2+) and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca(2+) is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca(2+) and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca(2+)/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca(2+) and CaM to influence eEF-2K activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase 2 stimulates osteoblast formation and inhibits osteoclast differentiation.

    PubMed

    Cary, Rachel L; Waddell, Seid; Racioppi, Luigi; Long, Fanxin; Novack, Deborah V; Voor, Michael J; Sankar, Uma

    2013-07-01

    Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. In vitro, although Camkk2⁻/⁻ mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2⁻/⁻ mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective, cell-permeable pharmacological inhibitor STO-609 also results in increased OB and diminished OC formation. Further, we find phospho-protein kinase A (PKA) and Ser¹³³ phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells, cytoplasmic (NFATc1). Moreover, in vivo administration of STO-609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)-induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and

  4. Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin*

    PubMed Central

    Kang, Seungwoo; Xu, Mingxuan; Cooper, Edward C.; Hoshi, Naoto

    2014-01-01

    M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex. PMID:24627475

  5. Channel-anchored protein kinase CK2 and protein phosphatase 1 reciprocally regulate KCNQ2-containing M-channels via phosphorylation of calmodulin.

    PubMed

    Kang, Seungwoo; Xu, Mingxuan; Cooper, Edward C; Hoshi, Naoto

    2014-04-18

    M-type potassium channels, encoded by the KCNQ family genes (KCNQ2-5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex.

  6. Reduced Arrhythmia Inducibility with Calcium/Calmodulin-Dependent Protein Kinase II Inhibition in Heart Failure Rabbits

    PubMed Central

    Hoeker, Gregory S.; Hanafy, Mohamed A.; Oster, Robert A.; Bers, Donald M.; Pogwizd, Steven M.

    2015-01-01

    Rationale Calcium/calmodulin-dependent protein kinase II (CaMKII) is activated in heart failure (HF) and can contribute to arrhythmias induced by β-adrenergic receptor-mediated sarcoplasmic reticulum calcium leak. Objective To evaluate the effect of CaMKII inhibition on ventricular tachycardia (VT) induction in conscious HF and naïve rabbits. Methods and Results Nonischemic HF was induced by aortic insufficiency and constriction. Electrocardiograms were recorded in rabbits pretreated with vehicle (saline) or the CaMKII inhibitor KN-93 (300 μg/kg); VT was induced by infusion of increasing doses of norepinephrine (NE, 1.56-25 μg/kg/min) in naïve (n = 8) and HF (n = 7) rabbits. With saline, median VT dose threshold in HF was 6.25 versus 12.5 μg/kg/min NE in naïve rabbits (p = 0.06). Pretreatment with KN-93 significantly increased VT threshold in HF and naïve rabbits (median = 25 μg/kg/min, p < 0.05 versus saline for both groups). Mean cycle length of VT initiation was shorter in HF (221 ± 20 ms) than naïve (296 ± 23 ms, p < 0.05) rabbits with saline; this difference was not significant after treatment with KN-93. Conclusions KN-93 significantly reduced arrhythmia inducibility and slowed initiation of VT, suggesting that CaMKII inhibition may have antiarrhythmic effects in the failing human heart. PMID:26650851

  7. Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3.

    PubMed

    Kramerova, I; Kudryashova, E; Ermolova, N; Saenz, A; Jaka, O; López de Munain, A; Spencer, M J

    2012-07-15

    Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.

  8. Calmodulin kinase II is required for fight or flight sinoatrial node physiology.

    PubMed

    Wu, Yuejin; Gao, Zhan; Chen, Biyi; Koval, Olha M; Singh, Madhu V; Guan, Xiaoqun; Hund, Thomas J; Kutschke, William; Sarma, Satyam; Grumbach, Isabella M; Wehrens, Xander H T; Mohler, Peter J; Song, Long-Sheng; Anderson, Mark E

    2009-04-07

    The best understood "fight or flight" mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by beta-adrenergic receptor (betaAR) agonist stimulation. HCN4 conducts an inward "pacemaker" current (I(f)) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I(f)-independent pathways are critical to SAN fight or flight responses. The multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) is a downstream signal in the betaAR pathway that activates Ca(2+) homeostatic proteins in ventricular myocardium. Mice with genetic, myocardial and SAN cell CaMKII inhibition have significantly slower HRs than controls during stress, leading us to hypothesize that CaMKII actions on SAN Ca(2+) homeostasis are critical for betaAR agonist responses in SAN. Here we show that CaMKII mediates ISO HR increases by targeting SAN cell Ca(2+) homeostasis. CaMKII inhibition prevents ISO effects on SAN Ca(2+) uptake and release from intracellular sarcoplasmic reticulum (SR) stores that are necessary for increasing DDR. CaMKII inhibition has no effect on the ISO response in SAN cells when SR Ca(2+) release is disabled and CaMKII inhibition is only effective at slowing HRs during betaAR stimulation. These studies show the tightly coupled, but previously unanticipated, relationship of CaMKII to the betaAR pathway in fight or flight physiology and establish CaMKII as a critical signaling molecule for physiological HR responses to catecholamines.

  9. Calmodulin kinase II–mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice

    PubMed Central

    Chelu, Mihail G.; Sarma, Satyam; Sood, Subeena; Wang, Sufen; van Oort, Ralph J.; Skapura, Darlene G.; Li, Na; Santonastasi, Marco; Müller, Frank Ulrich; Schmitz, Wilhelm; Schotten, Ulrich; Anderson, Mark E.; Valderrábano, Miguel; Dobrev, Dobromir; Wehrens, Xander H.T.

    2009-01-01

    Atrial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via “leaky” ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction. PMID:19603549

  10. The role of calcium and calcium/calmodulin-dependent kinases in skeletal muscle plasticity and mitochondrial biogenesis.

    PubMed

    Chin, Eva R

    2004-05-01

    Intracellular Ca(2+) plays an important role in skeletal muscle excitation-contraction coupling and also in excitation-transcription coupling. Activity-dependent alterations in muscle gene expression as a result of increased load (i.e. resistance or endurance training) or decreased activity (i.e. immobilization or injury) are tightly linked to the level of muscle excitation. Differential expression of genes in slow- and fast-twitch fibres is also dependent on fibre activation. Both these biological phenomena are, therefore, tightly linked to the amplitude and duration of the Ca(2+) transient, a signal decoded downstream by Ca(2+)-dependent transcriptional pathways. Evidence is mounting that the calcineurin-nuclear factor of activated T-cells pathway and the Ca(2+)/calmodulin-dependent kinases (CaMK) II and IV play important roles in regulating oxidative enzyme expression, mitochondrial biogenesis and expression of fibre-type specific myofibrillar proteins. CaMKII is known to decode frequency-dependent information and is activated during hypertrophic growth and endurance adaptations. Thus, it was hypothesized that CaMKII, and possibly CaMKIV, are down regulated during muscle atrophy and levels of expression of CaMKII alpha, -II beta, -II gamma and -IV were assessed in skeletal muscles from young, aged and denervated rats. The results indicate that CaMKII gamma, but not CaMKIIalpha or -beta, is up regulated in aged and denervated soleus muscle and that CaMKIV is absent in skeletal but not cardiac muscle. Whether CaMKII gamma up-regulation is part of the pathology of wasting or a result of some adaptational response to atrophy is not known. Future studies will be important in determining whether insights from the adaptational response of muscle to increased loads will provide pharmacological approaches for increasing muscle strength or endurance to counter muscle wasting.

  11. A brain-specific Ca sup 2+ /calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca sup 2+ signaling

    SciTech Connect

    Frangakis, M.V.; Ohmstede, C.A.; Sahyoun, N. )

    1991-06-15

    A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme {approximately} equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.

  12. Calcium/calmodulin-dependent protein kinase regulates the PINK1/Parkin and DJ-1 pathways of mitophagy during sepsis.

    PubMed

    Zhang, Xianghong; Yuan, Du; Sun, Qian; Xu, Li; Lee, Emma; Lewis, Anthony J; Zuckerbraun, Brian S; Rosengart, Matthew R

    2017-10-01

    During sepsis and shock states, mitochondrial dysfunction occurs. Consequently, adaptive mechanisms, such as fission, fusion, and mitophagy, are induced to eliminate damaged portions or entire dysfunctional mitochondria. The regulatory PINK1/Parkin and DJ-1 pathways are strongly induced by mitochondrial depolarization, although a direct link between loss of mitochondrial membrane potential (ΔΨ) and mitophagy has not been identified. Mitochondria also buffer Ca(2+), and their buffering capacity is dependent on ΔΨ Here, we characterize a role for calcium/calmodulin-dependent protein kinase (CaMK) I in the regulation of these mechanisms. Loss of ΔΨ with either pharmacologic depolarization or LPS leads to Ca(2+)-dependent mitochondrial recruitment and activation of CaMKI that precedes the colocalization of PINK1/Parkin and DJ-1. CaMKI is required and serves as both a PINK1 and Parkin kinase. The mechanisms operate in both immune and nonimmune cells and are induced in in vivo models of endotoxemia, sepsis, and hemorrhagic shock. These data support the idea that CaMKI links mitochondrial stress with the PINK1/Parkin and DJ-1 mechanisms of mitophagy.-Zhang, X., Yuan, D., Sun, Q., Xu, L., Lee, E., Lewis, A. J., Zuckerbraun, B. S., Rosengart, M. R. Calcium/calmodulin-dependent protein kinase regulates the PINK1/Parkin and DJ-1 pathways of mitophagy during sepsis. © FASEB.

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

    PubMed Central

    Shi, Juan; Geshi, Naomi; Takahashi, Shinichi; Kiyonaka, Shigeki; Ichikawa, Jun; Hu, Yaopeng; Mori, Yasuo; Ito, Yushi; Inoue, Ryuji

    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 cells by a muscarinic agonist carbachol (CCh; 100 μm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 μm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln855–Glu877) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr487 was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr487 (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr487 is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg8-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr487 may be 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. PMID

  14. Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

    PubMed Central

    Sag, Can M.; Wadsack, Daniel P.; Khabbazzadeh, Sepideh; Abesser, Marco; Grefe, Clemens; Neumann, Kay; Opiela, Marie-Kristin; Backs, Johannes; Olson, Eric N.; Brown, Joan Heller; Neef, Stefan; Maier, Sebastian K.G.; Maier, Lars S.

    2010-01-01

    Background Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)δC mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 μm−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in

  15. Conditioned taste aversion and Ca/calmodulin-dependent kinase II in the parabrachial nucleus of rats.

    PubMed

    Krivanek, J

    2001-07-01

    Bielavska and colleagues (Bielavska, Sacchetti, Baldi, & Tassoni, 1999) have recently shown that KN-62, an inhibitor of calcium/calmodulin-dependent kinase II (CaCMK), induces conditioned taste aversion (CTA) when introduced into the parabrachial nucleus (PBN) of rats. The aim of the present report was to assess whether activity of CaCMK in the PBN is changed during CTA. We induced CTA in one group of rats by pairing saccharin consumption with an ip injection of lithium chloride. Another group of rats received lithium alone (without being paired with saccharin consumption) to test whether lithium has an effect on CaCMK in the PBN, independent of those effects due to training. In animals receiving CTA training, CaCMK activity in extracts of PBN was reduced by approximately 30% at the postacquisition intervals of 12, 24, and 48 h, compared to control animals receiving saccharin with saline injection. By 120 h after CTA training, no effect on CaCMK was present. At those postacquisition intervals showing CaCMK activity effects due to CTA, there were no effects attributable to lithium alone. Lithium alone produced only a short-lasting reduction in CaCMK activity (at 20 min a 30% decrease, at 60 min a 23% decrease; and at 6, 12, and 24 h no decrease). The time course of lithium-induced effects differed markedly from that of CTA training. All changes were Ca2+/- -dependent; we did not observe any changes in Ca-independent activity. CTA effects on CaCMK were selective for PBN, insofar as we did not observe any CTA effects on CaCMK in the visual cortex, a brain region unrelated to taste pathways. Since CTA produces a relatively long-lasting reduction in CaCMK activity (lasting 2 days or more) specifically in the PBN, which is critical a relay for taste information, the reduction of CaCMK activity may enable the consolidation of taste memory in an aversive situation.

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

    NASA Technical Reports Server (NTRS)

    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.

  17. Calcium/calmodulin-dependent protein kinase IV mediates acute nicotine-induced antinociception in acute thermal pain tests.

    PubMed

    Jackson, Kia J; Damaj, Mohamad I

    2013-12-01

    Calcium-activated second messengers such as calcium/calmodulin-dependent protein kinase II have been implicated in drug-induced antinociception. The less abundant calcium-activated second messenger, calcium/calmodulin-dependent protein kinase IV (CaMKIV), mediates emotional responses to pain and tolerance to morphine analgesia but its role in nicotine-mediated antinociception is currently unknown. The goal of this study was to evaluate the role of CaMKIV in the acute effects of nicotine, primarily acute nicotine-induced antinociception. CaMKIV knockout (-/-), heterozygote (+/-), and wild-type (+/+) mice were injected with various doses of nicotine and evaluated in a battery of tests, including the tail-flick and hot-plate tests for antinociception, body temperature, and locomotor activity. Our results show a genotype-dependent reduction in tail-flick and hot-plate latency in CaMKIV (+/-) and (-/-) mice after acute nicotine treatment, whereas no difference was observed between genotypes in the body temperature and locomotor activity assessments. The results of this study support a role for CaMKIV in acute nicotine-induced spinal and supraspinal pain mechanisms, and further implicate involvement of calcium-dependent mechanisms in drug-induced antinociception.

  18. Calcium-calmodulin dependent protein kinase mediates the intracellular signaling pathways of cardiac apoptosis in mice with impaired glucose tolerance.

    PubMed

    Federico, Marilen; Portiansky, Enrique L; Sommese, Leandro; Alvarado, Francisco J; Blanco, Paula G; Zanuzzi, Carolina N; Dedman, John; Kaetzel, Marcia; Wehrens, Xander H T; Mattiazzi, Alicia; Palomeque, Julieta

    2017-01-20

    Background The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We described that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca(2+) /calmodulin-protein kinase (CaMKII) activity, ryanodine receptors (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca(2+) leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. Methods and Results We generated a pre-diabetic model in mice fed with FRD. FRD-mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca(2+) spontaneous events in the absence of SR Ca(2+) load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycemia significantly enhanced [(3) H]Ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycemia. CaMKII-inhibition prevented hyperglycemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the ROS scavenger Tempol prevented FRD-induced apoptosis in WT-mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodeling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. Conclusions CaMKII phosphorylation of RyR2, SR Ca(2+) leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favor Ca(2+) transit between both organelles. This article is protected by copyright. All rights reserved.

  19. Association of calcium/calmodulin-dependent protein kinase kinase1 rs7214723 polymorphism with lung cancer risk in a Chinese population.

    PubMed

    Chen, Da; Zhong, Fangming; Chen, Ye

    2017-07-24

    Calcium/calmodulin-dependent protein kinase kinase1 (CAMKK1) could specially recognize and activate Calcium/calmodulin-dependent protein kinase (CAMK) I and IV. Furthermore, The activation of CAMK showed positively correlation with proliferation of lung cancer (LC). In addition, A genome-wide association study has identified rs7214723 (E375G) in the CAMKK1 gene as a susceptibility locus for LC in the UK population. Therefore, we conducted a case-control study involving 320 LC patients and 320 controls to validate this conclusion in a Chinese population. Genotyping was performed using a custom-by-design 48-Plex single nucleotide polymorphism (SNP) Scan™ Kit. Our results indicate that the individuals with CC genotype of rs7214723 polymorphism had the higher risk of LC than those who carried TT genotype. Moreover, CAMKK1 rs7214723 polymorphism showed positively correlation with the elevated risk of LC in the allelic model and recessive model, but not in the dominant model. Stratified analysis further confirmed this significant association in male groups and smokers. In conclusion, CAMKK1 rs7214723 polymorphism may be associated with the increased risk of LC. However, larger studies with more diverse ethnic populations are needed to confirm these results. ©2017 The Author(s).

  20. Loss of Ca2+/calmodulin kinase kinase beta affects the formation of some, but not all, types of hippocampus-dependent long-term memory.

    PubMed

    Peters, Marco; Mizuno, Keiko; Ris, Laurence; Angelo, Marco; Godaux, Emile; Giese, K Peter

    2003-10-29

    Long-term memory (LTM) requires activation of the transcription factor cAMP-responsive element binding protein (CREB). Signaling by the Ca2+/calmodulin (CaM) kinase cascade has been implicated in CREB activation and memory consolidation processes in the hippocampus. The CaM kinase kinase beta isoforms belong to the CaM kinase cascade, and we have generated null mutant mice to investigate the role of these kinases in several forms of learning and memory. The null mutants were impaired in spatial training-induced CREB activation and spatial memory formation. Furthermore, the mutants lacked late, but not early, long-term potentiation at the hippocampal CA1 synapse, and they were impaired in LTM, but not short-term memory, for the social transmission of food preferences. We suggest that the CaM kinase kinasebeta isoforms are required for the formation of hippocampal LTM. Surprisingly, however, these kinases were not needed for contextual, trace fear, and passive avoidance LTM. Our results demonstrate that different signaling processes underlie the formation of these types of hippocampal LTM.

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

    PubMed

    Xu, Shucheng

    2010-09-01

    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 H(2)O(2) 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 H(2)O(2) not only induced the activation 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 H(2)O(2) 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 H(2)O(2) plays a pivotal role in ABA signaling. We infer that CaMK acts both upstream and downstream of H(2)O(2), but mainly acts between ABA and H(2)O(2) in ABA-induced antioxidant-defensive signaling.

  2. Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

    PubMed

    Atanasijevic, Tatjana; Shusteff, Maxim; Fam, Peter; Jasanoff, Alan

    2006-10-03

    We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

  3. Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer

    PubMed Central

    Parmer, T G; Ward, M D; Yurkow, E J; Vyas, V H; Kearney, T J; Hait, W N

    1999-01-01

    Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting, CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast

  4. Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin.

    PubMed

    White, James; Guerin, Theresa; Swanson, Hollie; Post, Steven; Zhu, Haining; Gong, Ming; Liu, Jun; Everson, William V; Li, Xiang-An; Graf, Gregory A; Ballard, Hubert O; Ross, Stuart A; Smart, Eric J

    2008-01-01

    In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

  5. Tau-Induced Ca(2+)/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

    PubMed

    Wei, Yu-Ping; Ye, Jin-Wang; Wang, Xiong; Zhu, Li-Ping; Hu, Qing-Hua; Wang, Qun; Ke, Dan; Tian, Qing; Wang, Jian-Zhi

    2017-06-23

    Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca(2+) concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca(2+) concentration with a simultaneous increase in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca(2+)/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca(2+)/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca(2+)/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca(2+) concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

  6. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

    PubMed Central

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells

  7. Activated Ca2+/calmodulin-dependent protein kinase IIgamma is a critical regulator of myeloid leukemia cell proliferation.

    PubMed

    Si, Jutong; Collins, Steven J

    2008-05-15

    Ca(2+) signaling is an important component of signal transduction pathways regulating B and T lymphocyte proliferation, but the functional role of Ca(2+) signaling in regulating myeloid leukemia cell proliferation has been largely unexplored. We observe that the activated (autophosphorylated) Ca(2+)/calmodulin-dependent protein kinase IIgamma (CaMKIIgamma) is invariably present in myeloid leukemia cell lines as well as in the majority of primary acute myelogenous leukemia patient samples. In contrast, myeloid leukemia cells induced to terminally differentiate or undergo growth arrest display a marked reduction in this CaMKIIgamma autophosphorylation. In cells harboring the bcr-abl oncogene, the activation (autophosphorylation) of CaMKIIgamma is regulated by this oncogene. Moreover, inhibition of CaMKIIgamma activity with pharmacologic agents, dominant-negative constructs, or short hairpin RNAs inhibits the proliferation of myeloid leukemia cells, and this is associated with the inactivation/down-regulation of multiple critical signal transduction networks involving the mitogen-activated protein kinase, Janus-activated kinase/signal transducers and activators of transcription (Jak/Stat), and glycogen synthase kinase (GSK3beta)/beta-catenin pathways. In myeloid leukemia cells, CaMKIIgamma directly phosphorylates Stat3 and enhances its transcriptional activity. Thus, CaMKIIgamma is a critical regulator of multiple signaling networks regulating the proliferation of myeloid leukemia cells. Inhibiting CaMKIIgamma may represent a novel approach in the targeted therapy of myeloid leukemia.

  8. Developmental iodine deficiency and hypothyroidism reduce phosphorylation of calcium/calmodulin-dependent kinase II in the rat entorhinal cortex.

    PubMed

    Wang, Yi; Hou, Yi; Dong, Jing; Xu, Hongde; Gong, Jian; Chen, Jie

    2010-12-01

    Iodine is essential for the synthesis of triiodothyronine (T₃) and thyroxine (T₄). Iodine deficiency leads to inadequate thyroid hormone. Hypothyroidism induced by iodine deficiency during gestation and postnatal period leads to cognitive deficits in learning and memory. However, the mechanism underlying these deficits is unclear. Calcium-dependent calmodulin kinase II (CaMKII) known as a potential memory molecule regulates important neuronal functions including learning and memory. Recent studies have shown that hypothyroidism alters phosphorylation of CaMKII in hippocampus or even in sympathetic ganglia of rats. Though the entorhinal cortex (EC) is an important functional structure within the neuronal network responsible for learning and memory, little is known about the effect of hypothyroidism on phosphorylation of CaMKII in the EC. Here, we report that iodine deficiency and propylthiouracil treatment through gestation and lactation reduce phosphorylation of CaMKII in the EC of pups. The increase of calcineurin, as well as reduction of neurogranin and calmodulin, may account for the reduced phosphorylation of CaMKII induced by developmental iodine deficiency and hypothyroidism. These findings in the EC may contribute to understanding the mechanisms that underlie impairment of learning and memory induced by developmental iodine deficiency and hypothyroidism.

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Gating of long-term depression by Ca2+/calmodulin-dependent protein kinase II through enhanced cGMP signalling in cerebellar Purkinje cells

    PubMed Central

    Kawaguchi, Shin-ya; Hirano, Tomoo

    2013-01-01

    Long-term depression (LTD) at parallel fibre synapses on a cerebellar Purkinje cell has been regarded as a cellular basis for motor learning. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the LTD induction as an important Ca2+-sensing molecule, the underlying signalling mechanism remains unclear. Here, we attempted to explore the potential signalling pathway underlying the CaMKII involvement in LTD using a systems biology approach, combined with validation by electrophysiological and FRET imaging experiments on a rat cultured Purkinje cell. Model simulation predicted the following cascade as a candidate mechanism for the CaMKII contribution to LTD: CaMKII negatively regulates phosphodiesterase 1 (PDE1), subsequently facilitates the cGMP/protein kinase G (PKG) signalling pathway and down-regulates protein phosphatase 2A (PP-2A), thus supporting the LTD-inducing positive feedback loop consisting of mutual activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This model suggestion was corroborated by whole-cell patch clamp recording experiments. In addition, FRET measurement of intracellular cGMP concentration revealed that CaMKII activation causes sustained increase of cGMP, supporting the signalling mechanism of LTD induction by CaMKII. Furthermore, we found that activation of the cGMP/PKG pathway by nitric oxide (NO) can support LTD induction without activation of CaMKII. Thus, this study clarified interaction between NO and Ca2+/CaMKII, two important factors required for LTD. PMID:23297306

  11. Activation of calcium- and calmodulin-dependent protein kinase (CCaMK), the central regulator of plant root endosymbiosis.

    PubMed

    Singh, Sylvia; Parniske, Martin

    2012-08-01

    The key molecular event during the development of arbuscular mycorrhiza and the root nodule symbiosis is the activation of calcium- and calmodulin-dependent protein kinase (CCaMK). Its regulation is complex and involves positive as well as negative regulation facilitated by autophosphorylation of two conserved sites. Deregulated versions of CCaMK are sufficient for mediating both organogenesis and infection processes. Epistasis tests demonstrated that a main function of signaling components upstream of calcium spiking is the activation of CCaMK. Despite CCaMK being a central signaling hub, specificity for both symbioses exists, resulting in differential transcriptional gene expression patterns. While the specificity upstream of CCaMK can be conceptualized by the specific perception of rhizobial and fungal lipo-chitooligosaccharides via cognate LysM receptors, the mechanisms conferring transcriptional specificity downstream of CCaMK are likely conferred by a variety of transcriptional regulators, mediating symbiosis appropriate gene regulation.

  12. Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Olson, N J; Massé, T; Suzuki, T; Chen, J; Alam, D; Kelly, P T

    1995-01-01

    To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element. Images Fig. 1 Fig. 3 PMID:7878035

  13. Ca(2+)/calmodulin-dependent protein kinase mediates the phosphorylation of CD44 required for cell migration on hyaluronan.

    PubMed Central

    Lewis, C A; Townsend, P A; Isacke, C M

    2001-01-01

    CD44 is the principal cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and binding to this ligand underlies CD44-mediated cell attachment and migration. As would be expected for a widely expressed adhesion receptor, CD44 is subject to complex regulatory events, and mis-regulation of the receptor has been associated with a number of disease pathologies, including chronic inflammatory conditions and the progression of metastatic tumours. In previous studies we have demonstrated that a key control point for this receptor is the phosphorylation of CD44 on a conserved cytoplasmic serine residue, Ser(325). This modification is not required for efficient ligand binding, but is an essential component of CD44-dependent cell migration on a hyaluronan substratum. To understand better the mechanism regulating CD44 phosphorylation on Ser(325), we have generated a monoclonal antibody that specifically recognizes CD44 phosphorylated on Ser(325), and have developed assays to identify the Ser(325) kinase. We demonstrate here that CD44 is phosphorylated to high stoichiometry in resting cells and that Ca(2+)/calmodulin-dependent protein kinase II is a CD44 Ser(325) kinase. PMID:11463356

  14. Crystal structure of the Ca²⁺/calmodulin-dependent protein kinase kinase in complex with the inhibitor STO-609.

    PubMed

    Kukimoto-Niino, Mutsuko; Yoshikawa, Seiko; Takagi, Tetsuo; Ohsawa, Noboru; Tomabechi, Yuri; Terada, Takaho; Shirouzu, Mikako; Suzuki, Atsushi; Lee, Suni; Yamauchi, Toshimasa; Okada-Iwabu, Miki; Iwabu, Masato; Kadowaki, Takashi; Minokoshi, Yasuhiko; Yokoyama, Shigeyuki

    2011-06-24

    Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca(2+) elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro(274), which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases.

  15. Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    SciTech Connect

    Gangopadhyay, Jaya Pal; Ikemoto, Noriaki

    2010-05-28

    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 Ca{sup 2+} 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 Ca{sup 2+}/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 Ca{sup 2+} 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 Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} 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 Ca{sup 2+} 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

  16. Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca sup 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Gandy, S.; Czernik, A.J.; Greengard, P. )

    1988-08-01

    The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggest a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP. Ca{sup 2+}/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. Using rat cerebral cortex synaptosomes prelabeled with {sup 32}P{sub i}, a {sup 32}P-labeled phosphoprotein of {approx}135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.

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

    NASA Technical Reports Server (NTRS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  19. 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)

    SciTech Connect

    Onouchi, Takashi; Sueyoshi, Noriyuki; Ishida, Atsuhiko; Kameshita, Isamu

    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.

  20. Attenuation of the seizure-induced expression of BDNF mRNA in adult rat brain by an inhibitor of calcium/calmodulin-dependent protein kinases.

    PubMed

    Murray, K D; Hayes, V Y; Gall, C M; Isackson, P J

    1998-01-01

    We have examined the potential involvement of calcium/calmodulin-dependent protein kinases in the regulation of brain-derived neurotrophic factor mRNA in vivo following kainic acid (kainate)-induced seizure activity by in situ hybridization. KN-62, a specific inhibitor of calcium/calmodulin-dependent protein kinase type II and IV, blocked the characteristic induction of brain-derived neurotrophic factor mRNA seen following seizure activity. This blockade was specific to calcium/calmodulin-dependent protein kinase type II and IV as inhibitors of both protein kinase C and cAMP-dependent protein kinase had no effect. Inhibition of brain-derived neurotrophic factor mRNA increases varied between brain regions; an almost complete inhibition was seen throughout cortical regions, whereas only partial inhibitory effects were noted within hippocampus. A similar inhibition of increased c-fos mRNA was observed throughout cortical, hippocampal and diencephalic regions. The two predominant brain-derived neurotrophic factor transcripts induced by kainate, containing exons I or III, were differentially affected by KN-62. The cortical induction of exon I was blocked by KN-62, whereas exon III was not, providing additional evidence for the differential regulation of individual brain-derived neurotrophic factor transcripts and demonstrating that inhibition of brain-derived neurotrophic factor induction was not due to general blockade of seizure activity throughout the neocortex. These data implicate calcium/calmodulin-dependent protein kinase type II or IV in the regulation of brain-derived neurotrophic factor mRNA in vivo and suggest regionally specific mechanisms occur throughout the brain.

  1. Tertiary Structural Rearrangements upon Oxidation of Methionine145 in Calmodulin Promotes Targeted Proteasomal Degradation

    SciTech Connect

    Sacksteder, Colette A.; Whittier, Jennifer E.; Xiong, Yijia; Li, Jinhui; Galeva, Nadezhda A.; Jacoby, Michael E.; Purvine, Samuel O.; Williams, Todd D.; Rechsteiner, Martin C.; Bigelow, Diana J.; Squier, Thomas C.

    2006-08-15

    Mass spectrometry was used to identify possible linkages between the oxidation of specific methionines in calmodulin (CaM) and its degradation by the proteasome. Degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large fragment (Q41-D131) and a carboxyl-terminus peptide (G132-K148) enriched in methionine sulfoxide [Met(O)] prior to rebinding and complete digestion. Rates of CaM degradation correlate with the oxidation of Met145, which is preferentially oxidized by hydrogen peroxide prior to the oxidation of Met144. The specificity of CaMox degradation was confirmed using CaM mutants in which the majority of methionines were replaced by leucines using site-directed mutagenesis, permitting the site-specific oxidation of Met144 or Met145. Oxidation of both Met144 and Met145 results in the selective degradation of CaMox by the proteasome with rates comparable to that observed for fully oxidized CaM. Oxidation of Met144 has little effect on proteolytic degradation; rather, oxidation of Met145 targets CaMox for proteasomal degradation. The selective oxidation of either Met144 or Met145 has little effect on the secondary structure of CaM; rather, oxidation-induced tertiary structural changes mediate the recognition and degradation of CaMox by the proteasome. Thus, oxidation of Met145 can function as a sensor that has the potential to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.

  2. Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

    NASA Technical Reports Server (NTRS)

    Zhang, J. Z.; Wu, Y.; Williams, B. Y.; Rodney, G.; Mandel, F.; Strasburg, G. M.; Hamilton, S. L.

    1999-01-01

    This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of 125I-labeled CaM at both micromolar and nanomolar Ca2+ concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free 125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

  3. Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

    NASA Technical Reports Server (NTRS)

    Zhang, J. Z.; Wu, Y.; Williams, B. Y.; Rodney, G.; Mandel, F.; Strasburg, G. M.; Hamilton, S. L.

    1999-01-01

    This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of 125I-labeled CaM at both micromolar and nanomolar Ca2+ concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free 125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

  4. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    PubMed

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  5. Phosphorylation of CEACAM1 Molecule by Calmodulin Kinase IID in a Three-dimensional Model of Mammary Gland Lumen Formation*

    PubMed Central

    Nguyen, Tung; Chen, Charng-Jui; Shively, John E.

    2014-01-01

    Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), a transmembrane protein, expressed on normal breast epithelial cells is down-regulated in breast cancer. Phosphorylation of Thr-457 on the short cytoplasmic domain isoform (CEACAM1-SF) that is predominant in normal epithelial cells is required for lumen formation in a three-dimensional model that involves apoptosis of the central acinar cells. Calmodulin kinase IID (CaMKIID) was selected as a candidate for the kinase required for Thr-457 phosphorylation from a gene chip analysis comparing genes up-regulated in MCF7 cells expressing wild type CEACAM1-SF compared with the T457A-mutated gene (Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749–5760). Up-regulation of CaMKIID during lumen formation was confirmed by analysis of mRNA and protein levels. CaMKIID was able to phosphorylate a synthetic peptide corresponding to the cytoplasmic domain of CEACAM1-SF and was covalently bound to biotinylated and T457C-modified peptide in the presence of a kinase trap previously described by Shokat and co-workers (Maly, D. J., Allen, J. A., and Shokat, K. M. (2004) J. Am. Chem. Soc. 126, 9160–9161). When cell lysates from wild type-transfected MCF7 cells undergoing lumen formation were incubated with the peptide and kinase trap, a cross-linked band corresponding to CaMKIID was observed. When these cells were treated with an RNAi that inhibits CaMKIID expression, lumen formation was blocked by over 90%. We conclude that CaMKIID specifically phosphorylates Thr-457 on CEACAM1-SF, which in turn regulates the process of lumen formation via apoptosis of the central acinar cells. PMID:24302721

  6. The Octopamine Receptor OAMB Mediates Ovulation via Ca2+/Calmodulin-Dependent Protein Kinase II in the Drosophila Oviduct Epithelium

    PubMed Central

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  7. Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

    PubMed

    Arrázola, Macarena S; Varela-Nallar, Lorena; Colombres, Marcela; Toledo, Enrique M; Cruzat, Fernando; Pavez, Leonardo; Assar, Rodrigo; Aravena, Andrés; González, Mauricio; Montecino, Martín; Maass, Alejandro; Martínez, Servet; Inestrosa, Nibaldo C

    2009-12-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

  8. Activation of Ca2+-calmodulin kinase II induces desensitization by background light in dogfish retinal ‘on’ bipolar cells

    PubMed Central

    Shiells, R A; Falk, G

    2000-01-01

    Retinal ‘on’ bipolar cells possess a metabotropic glutamate receptor (mGluR6) linked to the control of a G-protein and cGMP-activated channels which functions to generate high synaptic amplification of rod signals under dark-adapted conditions. Desensitization of ‘on’ bipolar cells is initiated by a rise in Ca2+ during background light too weak to adapt rod photoreceptors. Desensitization could also be elicited by raising intracellular Ca2+ above 1 μm. In order to investigate the mechanism of desensitization, whole-cell current responses to brief flashes and to steps of light were obtained from voltage-clamped ‘on’ bipolar cells in dark-adapted dogfish retinal slices. The inclusion of Ca2+-calmodulin kinase II (CaMKII) inhibitor peptides in the patch pipette solutions not only blocked desensitization of ‘on’ bipolar cells by dim background light and by 50 μm Ca2+, but also increased their flash sensitivity. The substrate of phosphorylation by CaMKII is the ‘on’ bipolar cell cGMP-activated channels. Desensitization probably results from a reduction in their sensitivity to cGMP and a voltage-dependent decrease in their conductance. A role for protein kinase C (PKC) in this process was excluded since activating PKC independently of Ca2+ with the phorbol ester PMA failed to induce desensitization of ‘on’ bipolar cells. PMID:11034622

  9. Neuronal calcium/calmodulin-dependent protein kinase II mediates nicotine reward in the conditioned place preference test in mice.

    PubMed

    Jackson, Kia J; Muldoon, Pretal P; Walters, Carrie; Damaj, Mohamad Imad

    2016-02-01

    Several recent studies have indicated the involvement of calcium-dependent mechanisms, in particular the abundant calcium-activated kinase, calcium/calmodulin-dependent kinase II (CaMKII), in behaviors associated with nicotine dependence in mice. Behavioral and biochemical studies have shown that CaMKII is involved in acute and chronic nicotine behaviors and nicotine withdrawal; however, evidence of a role for CaMKII in nicotine reward is lacking. Thus, the goal of the current study was to examine the role of CaMKII in nicotine reward. Using pharmacological and genetic tools, we tested nicotine conditioned place preference (CPP) in C57Bl/6 mice after administration of CaMKII antagonists and in α-CaMKII wild-type (+/+) and heterozygote (±) mice. CaMKII antagonists blocked expression of nicotine CPP, and the preference score was significantly reduced in α-CaMKII ± mice compared with their +/+ counterparts. Further, we assessed CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex, and hippocampus after nicotine CPP and found significant increases in CaMKII activity in the mouse VTA and NAc that were blocked by CaMKII antagonists. The findings from this study show that CaMKII mediates nicotine reward and suggest that increases in CaMKII activity in the VTA and NAc are relevant to nicotine reward behaviors.

  10. Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.

    PubMed Central

    Butler, L S; Silva, A J; Abeliovich, A; Watanabe, Y; Tonegawa, S; McNamara, J O

    1995-01-01

    Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory. Images Fig. 2 PMID:7624331

  11. Urea-induced denaturation of human calcium/calmodulin-dependent protein kinase IV: a combined spectroscopic and MD simulation studies.

    PubMed

    Naz, Huma; Shahbaaz, Mohd; Haque, Md Anzarul; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2017-02-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme which belongs to the Ser/Thr kinase family. CaMKIV plays important role in varieties of biological processes such as gene expression regulation, memory consolidation, bone growth, T-cell maturation, sperm motility, regulation of microtubule dynamics, cell-cycle progression, and apoptosis. To measure stability parameters, urea-induced denaturation of CaMKIV was carried out at pH 7.4 and 25°C, using three different probes, namely far-UV CD, near-UV absorption, and tryptophan fluorescence. A coincidence of normalized denaturation curves of these optical properties suggests that urea-induced denaturation is a two-state process. Analysis of these denaturation curves gave values of 4.20 ± 0.12 kcal mol(-1), 2.95 ± 0.15 M, and 1.42 ± 0.06 kcal mol(-1) M(-1) for [Formula: see text] (Gibbs free energy change (ΔGD) in the absence of urea), Cm (molar urea concentration ([urea]) at the midpoint of the denaturation curve), and m (=∂ΔGD/∂[urea]), respectively. All these experimental observations have been fully supported by 30 ns molecular dynamics simulation studies.

  12. Type II CaS /calmodulin-dependent kinase phosphorylates tau protein in the region of the mouse repeat

    SciTech Connect

    Kosik, K.S.; Lee, G.; Kennedy, M.B.

    1987-05-01

    Tau is a phosphoprotein associated with the subset of microtubules present in the axonal domain of neurons and is a component of the Alzheimer neurofibrillary tangle. Tau protein was purified from bovine brain by the taxol method followed by gel filtration of the heat-stable microtubule fraction CaS /calmodulin-dependent kinase was purified from rat brain as previously described. Incubation of tau in the kinase-containing reaction mixture resulted in intense incorporation of TSP into the protein. Labeled tau protein was trypsinized and separated into discrete fragments by reverse-phase HPLC. The chromatogram contained two radioactive peaks, sequenced on a gas phase sequenator. The first peak was eluted into two tubes, with more radioactivity in the second tube. The sequence in the second tube contained 16 residues which corresponded to an identical sequence in mouse tau that is part of a longer stretch which repeats three times with greater than 50% homology. The highest CPM's were located on a PTH-conjugated threonine. The first tube contained the identical amino acid sequence and a lower level of radioactivity throughout unassociated with any single amino acid. About 1/3 of the total peptide was phosphorylated. A second radioactive peak contained an unidentifiable PTH-amino acid present at a level below the yield obtained for tau.

  13. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-d-aspartate receptor

    PubMed Central

    Leonard, A. Soren; Lim, Indra A.; Hemsworth, Daniel E.; Horne, Mary C.; Hell, Johannes W.

    1999-01-01

    The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca2+ influx through postsynaptic N-methyl-d-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIα by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca2+ influx but also close to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP. PMID:10077668

  14. Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize.

    PubMed

    Zhu, Yuan; Yan, Jingwei; Liu, Weijuan; Liu, Lei; Sheng, Yu; Sun, Yue; Li, Yanyun; Scheller, Henrik Vibe; Jiang, Mingyi; Hou, Xilin; Ni, Lan; Zhang, Aying

    2016-07-01

    Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize (Zea mays), which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 displays a partially overlapping expression pattern with ZmCCaMK after ABA treatment, and H2O2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates Ser-113 of ZmNAC84 in vitro, and Ser-113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco (Nicotiana tabacum) can improve drought tolerance and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H2O2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK. Subsequently, the activated ZmCCaMK phosphorylates ZmNAC84 at Ser-113, thereby inducing antioxidant defense by activating downstream genes.

  15. Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation.

    PubMed

    House, Suzanne J; Ginnan, Roman G; Armstrong, Shayn E; Singer, Harold A

    2007-06-01

    There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

  16. Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

    PubMed

    Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

    2016-11-01

    Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

  17. Effects of PU.1-induced mouse calcium-calmodulin-dependent kinase I-like kinase (CKLiK) on apoptosis of murine erythroleukemia cells.

    PubMed

    Yamada, Toshiyuki; Suzuki, Mitsuhiro; Satoh, Hitoshi; Kihara-Negishi, Fumiko; Nakano, Hiroyasu; Oikawa, Tsuneyuki

    2004-03-10

    PU.1, a hematopoietic cell-specific Ets family transcription factor, is involved in the generation of murine erythroleukemia (MEL). To identify the target gene(s) of PU.1 in MEL cells, we carried out differential display (DD) analysis and isolated a novel gene whose expression was up-regulated after overexpression of PU.1 in MEL cells. Because the gene exhibited about 90% homology with the human calcium-calmodulin-dependent kinase I-like kinase (CKLiK) gene, it was identified as a mouse homologue of human CKLiK. The mCKLiK gene was mapped to the mouse chromosome 2A1-A3 region and shown to be expressed predominantly in T cells lymphoma and embryonal carcinoma cell lines and primary thymus and brain. Two types of transcripts were present showing a difference in the 3' portion of the coding region and CREB-activating ability. Overexpression of each isoform of mCKLiK in MEL cells revealed that one of them induces, while the other inhibits apoptosis under low serum condition. Differentiation inhibition and lineage switch to myelomonocytes, which were previously observed in MEL cells overexpressing PU.1, were not provoked in the cells overexpressing mCKLiK. These results suggest that mCKLiK is up-regulated by PU.1 in MEL cells and involved in apoptosis of the cells.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity.

    PubMed

    Kumrungsee, Thanutchaporn; Akiyama, Sayaka; Guo, Jian; Tanaka, Mitsuru; Matsui, Toshiro

    2016-12-15

    Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca(2+)-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1μM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300μM Trp-Val or Trp-Ile. On the basis of Ca(2+)-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca(2+)-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6μM, respectively.

  20. Ocular dominance plasticity is stably maintained in the absence of alpha calcium calmodulin kinase II (alphaCaMKII) autophosphorylation.

    PubMed

    Taha, Sharif A; Stryker, Michael P

    2005-11-08

    The molecule alpha calcium calmodulin kinase II (alphaCaMKII) is known to play a fundamental role in the induction of many forms of synaptic plasticity. A major theory of alphaCaMKII 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 alphaCaMKII. These mutant mice are deficient in plasticity after monocular deprivation, but a sufficiently long period of monocular deprivation will induce ocular dominance plasticity. After induction of ocular dominance plasticity, the stability of the induced changes was assayed after binocular deprivation. Plasticity in homozygous mutant animals was as stable as that measured in WT littermates; also, response characteristics did not differ between the two groups. Our results suggest that alphaCaMKII autophosphorylation is required for the induction of ocular dominance plasticity but is not needed for its stable maintenance thereafter.

  1. Intracellular Ca2+ and Ca2+/Calmodulin-Dependent Kinase II Mediate Acute Potentiation of Neurotransmitter Release by Neurotrophin-3

    PubMed Central

    He, Xiang-ping; Yang, Feng; Xie, Zuo-ping; Lu, Bai

    2000-01-01

    Neurotrophins have been shown to acutely modulate synaptic transmission in a variety of systems, but the underlying signaling mechanisms remain unclear. Here we provide evidence for an unusual mechanism that mediates synaptic potentiation at the neuromuscular junction (NMJ) induced by neurotrophin-3 (NT3), using Xenopus nerve–muscle co-culture. Unlike brain-derived neurotrophic factor (BDNF), which requires Ca2+ influx for its acute effect, NT3 rapidly enhances spontaneous transmitter release at the developing NMJ even when Ca2+ influx is completely blocked, suggesting that the NT3 effect is independent of extracellular Ca2+. Depletion of intracellular Ca2+ stores, or blockade of inositol 1, 4, 5-trisphosphate (IP3) or ryanodine receptors, prevents the NT3-induced synaptic potentiation. Blockade of IP3 receptors can not prevent BDNF-induced potentiation, suggesting that BDNF and NT3 use different mechanisms to potentiate transmitter release. Inhibition of Ca2+/calmodulin-dependent kinase II (CaMKII) completely blocks the acute effect of NT3. Furthermore, the NT3-induced potentiation requires a continuous activation of CaMKII, because application of the CaMKII inhibitor KN62 reverses the previously established NT3 effect. Thus, NT3 potentiates neurotransmitter secretion by stimulating Ca2+ release from intracellular stores through IP3 and/or ryanodine receptors, leading to an activation of CaMKII. PMID:10811820

  2. Calcium/calmodulin-dependent protein kinase IIalpha in optic axons moves with slow axonal transport and undergoes posttranslational modification.

    PubMed

    Lund, L M; McQuarrie, I G

    2001-12-21

    In neurons, the mRNA for calcium/calmodulin-dependent protein kinase II alpha (CKIIalpha) is known to be targeted to dendrites-where the enzyme is synthesized and supports postsynaptic functions. We are interested in knowing how neuronal proteins enter axons from the nerve cell body, and the mechanism for protein transport to terminals. Because CKIIalpha immunofluorescence can be demonstrated in over 80% of retinal ganglion cells, we asked whether this regulatory protein is being transported into optic axons. Using Sprague-Dawley rats, [(35)S] methionine was injected into the vitreous humor of the eye. Four days later, the optic nerves, tracts, lateral geniculate ganglia, and superior colliculi were removed and processed for 2D-PAGE and Western blotting. Radiolabeled CKIIalpha appears to move with slow component b (SCb) of axonal transport, as is the case in rodent sciatic motor neurons. In addition, the radiolabeled CKIIalpha isoform that enters the optic nerve is found to be 4 kDa heavier (in SDS-PAGE molecular mass) than the isoform in the optic tract, superior colliculus, and lateral geniculate nucleus. This reduction is likely the result of dephosphorylation, which is a mechanism used to regulate the enzyme's activity.

  3. Resveratrol Inhibits Neuronal Apoptosis and Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Diabetic Mouse Retina

    PubMed Central

    Kim, Young-Hee; Kim, Yoon-Sook; Kang, Sang-Soo; Cho, Gyeong-Jae; Choi, Wan-Sung

    2010-01-01

    OBJECTIVE This study investigated the effects of resveratrol, a natural polyphenol with neuroprotective properties, on retinal neuronal cell death mediated by diabetes-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). RESEARCH DESIGN AND METHODS Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received buffer. All mice were killed 2 months after the injections, and the extent of neuronal cell death, CaMKII, and phospho-CaMKII protein expression levels and CaMKII kinase activity were examined in the retinas. To assess the role of CaMKII in the death of retinal neurons, a small-interfering RNA (siRNA) or specific inhibitor of CaMKII was injected into the right vitreous humor, and vehicle only was injected into the left vitreous humor, 2 days before death. Resveratrol (20 mg/kg) was administered by oral gavage daily for 4 weeks, beginning 1 month after the fifth injection of either STZ or buffer. RESULTS The death of retinal ganglion cells (RGCs), CaMKII, phospho-CaMKII protein levels, and CaMKII activity were all greatly increased in the retinas of diabetic mice compared with controls, 2 months after induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive signals co-localized with CaMKII- and phospho-CaMKII immunoreactive RGCs. However, in addition to CaMKII knockdown and inhibition by siRNA or a specific inhibitor, respectively, resveratrol provided complete protection from diabetes-induced retinal cell death. CONCLUSIONS In the present study, resveratrol prevented diabetes-induced RGC death via CaMKII downregulation, implying that resveratrol may have potential therapeutic applications for prevention of diabetes-induced visual dysfunction. PMID:20424226

  4. Curcumin Attenuates Opioid Tolerance and Dependence by Inhibiting Ca2+/Calmodulin-Dependent Protein Kinase II α Activity

    PubMed Central

    Hu, Xiaoyu; Huang, Fang; Szymusiak, Magdalena

    2015-01-01

    Chronic use of opioid analgesics has been hindered by the development of opioid addiction and tolerance. We have reported that curcumin, a natural flavonoid from the rhizome of Curcuma longa, attenuated opioid tolerance, although the underlying mechanism remains unclear. In this study, we tested the hypothesis that curcumin may inhibit Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα), a protein kinase that has been previously proposed to be critical for opioid tolerance and dependence. In this study, we used state-of-the-art polymeric formulation technology to produce poly(lactic-co-glycolic acid) (PLGA)-curcumin nanoparticles (nanocurcumin) to overcome the drug’s poor solubility and bioavailability, which has made it extremely difficult for studying in vivo pharmacological actions of curcumin. We found that PLGA-curcumin nanoparticles reduced the dose requirement by 11- to 33-fold. Pretreatment with PLGA-curcumin (by mouth) prevented the development of opioid tolerance and dependence in a dose-dependent manner, with ED50 values of 3.9 and 3.2 mg/kg, respectively. PLGA-curcumin dose-dependently attenuated already-established opioid tolerance (ED50 = 12.6 mg/kg p.o.) and dependence (ED50 = 3.1 mg/kg p.o.). Curcumin or PLGA-curcumin did not produce antinociception by itself or affect morphine (1–10 mg/kg) antinociception. Moreover, we found that the behavioral effects of curcumin on opioid tolerance and dependence correlated with its inhibition of morphine-induced CaMKIIα activation in the brain. These results suggest that curcumin may attenuate opioid tolerance and dependence by suppressing CaMKIIα activity. PMID:25515789

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

    NASA Technical Reports Server (NTRS)

    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.

  6. Selective Nitration of Tyr(99) in Calmodulin as a Marker of Cellular Conditions of Oxidative Stress

    SciTech Connect

    Smallwood, Heather S. ); Galeva, Nadezhda A.; Bartlett, Ryan K.; Urbauer, Ramona J.; Williams, Todd D.; Urbauer, Jeffrey L.; Squier, Thomas C. )

    2003-01-01

    We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite(ONOO-)was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr-99 at physiological pH resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr-99 is readily nitrated by perioxynitrite and that methionine side chains do not function as an antioxidant in scavenging perioxynitrite. Thus, separate reactive species are involved in the oxidation of

  7. Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis.

    PubMed

    Tsaadon, Lina; Kaplan-Kraicer, Ruth; Shalgi, Ruth

    2008-05-01

    Sperm-egg fusion induces cortical granules exocytosis (CGE), a process that ensures the block to polyspermy. CGE can be induced independently by either a rise in intracellular calcium concentration or protein kinase C (PKC) activation. We have previously shown that myristoylated alanine-rich C kinase substrate (MARCKS) cross-links filamentous actin (F-actin) and regulates its reorganization. This activity is reduced either by PKC-induced MARCKS phosphorylation (PKC pathway) or by its direct binding to calmodulin (CaM; CaM pathway), both inducing MARCKS translocation, F-actin reorganization, and CGE. Currently, we examine the involvement of Ca(2)(+)/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca(2)(+)/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active alphaCaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt alphaCaMKII. Microinjecting eggs with mutant-unphosphorylatable MARCKS reduced the intensity of 12-O-tetradecanoylphorbol 13-acetate or ionomycin-induced CGE by 50%, indicating that phosphorylation of MARCKS by novel and/or conventional PKCs (n/cPKCs) is a pivotal event associated with CGE. Moreover, we were able to demonstrate cPKCs involvement in ionomycin-induced MARCKS translocation and CGE. These results led us to propose that MARCKS, rather than CaMKII, as a key mediator of CGE.

  8. Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.

    PubMed

    Marganski, William A; Gangopadhyay, Samudra S; Je, Hyun-Dong; Gallant, Cynthia; Morgan, Kathleen G

    2005-09-16

    Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.

  9. Loss of the Calmodulin-Dependent Inhibition of RyR1 Calcium Release Channel Upon Oxidation of Methionines in Calmodulin

    SciTech Connect

    Boschek, Curt B.; Jones, Terry E.; Smallwood, Heather S.; Squier, Thomas C.; Bigelow, Diana J.

    2008-01-08

    The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its affect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of 3[H]-ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, where calcium-activated CaM acts to enhance the calcium-sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially diminish the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in a minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response to enhance the duration of calcium transients to promote enhanced

  10. Loss of the calmodulin-dependent inhibition of the RyR1 calcium release channel upon oxidation of methionines in calmodulin.

    PubMed

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

    2008-01-08

    The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of [3H]ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, in which calcium-activated CaM acts to enhance the calcium sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially weaken the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not significantly affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as part of a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress observed during biological aging. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response

  11. Involvement of Amygdalar Protein Kinase A, but not Calcium/Calmodulin-Dependent Protein Kinase II, in the Reconsolidation of Cocaine-Related Contextual Memories in Rats

    PubMed Central

    Arguello, Amy A.; Hodges, Matthew A.; Wells, Audrey M.; Lara, Honorio; Xie, Xiaohu; Fuchs, Rita A.

    2013-01-01

    Rationale Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response-cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon. Objectives The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response-cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior. Methods Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context. Results Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion. Conclusions PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental

  12. Gene Expression Profile of Calcium/Calmodulin-Dependent Protein Kinase IIα in Rat's Hippocampus during Morphine Withdrawal

    PubMed Central

    Ahmadi, Shamseddin; Amiri, Shahin; Rafieenia, Fatemeh; Rostamzadeh, Jalal

    2013-01-01

    Introduction Calcium/calmodulin-dependent protein kinase II (CaMKII) which is highly expressed in the hippocampus is known to play a pivotal role in reward-related memories and morphine dependence. Methods In the present study, repeated morphine injections once daily for 7 days was done to induce morphine tolerance in male Wistar rats, after which gene expression profile of α-isoform of CaMKII (CaMKIIα) in the hippocampus was evaluated upon discontinuation of morphine injection over 21 days of morphine withdrawal. Control groups received saline for 7 consecutive days. For gene expression study, rats’ brains were removed and the hippocampus was dissected in separate groups on days 1, 3, 7, 14, and 21 since discontinuation of of morphine injection. A semi-quantitative RT-PCR method was used to evaluate the gene expression profile. Results Tolerance to morphine was verified by a significant decrease in morphine analgesia in a hotplate test on day 8 (one day after the final repeated morphine injections). Results showed that gene expression of CaMKIIα at mRNA level on day 1, 3, 7, 14 and 21 of morphine withdrawal was significantly altered as compared to the saline control group. Post hoc Tukey's test revealed a significantly enhanced CaMKIIα gene expression on day 14. Discussion It can be concluded that CaMKIIα gene expression during repeated injections of morphine is increased and this increase continues up to 14 days of withdrawal then settles at a new set point. Therefore, the strong morphine reward-related memory in morphine abstinent animals may, at least partly be attributed to, the up-regulation of CaMKIIα in the hippocampus over 14 days of morphine withdrawal. PMID:25337341

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

    PubMed

    Jackson, Kia J; Sanjakdar, Sarah S; Chen, Xiangning; Damaj, M Imad

    2012-01-01

    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.

  14. MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ)

    PubMed Central

    Liu, Yong Feng; Spinelli, Amy; Sun, Li-Yan; Jiang, Miao; Singer, Diane V.; Ginnan, Roman; Saddouk, Fatima Z.; Van Riper, Dee; Singer, Harold A.

    2016-01-01

    The multifunctional Ca2+/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3′UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis. PMID:27199283

  15. MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ).

    PubMed

    Liu, Yong Feng; Spinelli, Amy; Sun, Li-Yan; Jiang, Miao; Singer, Diane V; Ginnan, Roman; Saddouk, Fatima Z; Van Riper, Dee; Singer, Harold A

    2016-05-20

    The multifunctional Ca(2+)/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3'UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis.

  16. Diabetes mellitus affects activity of calcium/calmodulin-dependent protein kinase II alpha in rat trigeminal ganglia.

    PubMed

    Jerić, Milka; Vuica, Ana; Borić, Matija; Puljak, Livia; Jeličić Kadić, Antonia; Grković, Ivica; Filipović, Natalija

    2015-01-01

    The activity of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The aim of this study was to investigate the immunoreactivity of phosphorylated CaMKIIα (pCaMKIIα) in subpopulations of trigeminal ganglion (TG) neurons in rat models of early diabetes type 1 (dm1) and 2 (dm2). DM1 model was induced with intraperitoneally (i.p.) injected streptozotocin (STZ) (55mg/kg). DM2 rats were fed with the high fat diet (HFD) for 2 weeks and then received 35mg/kg of STZ i.p. Two weeks and 2 months after the STZ-diabetes induction, rats were sacrificed and immunohistochemical analysis for detection of pCaMKIIα immunoreactivity and double immunofluorescence labelling with isolectin (IB4) was performed. Increased intensity of pCaMKIIα immunofluorescence, restricted to IB4-negative small-diameter neurons, was seen in TG neurons two months after STZ-DM1 induction. DM1 model, as well as the obesity (control dm2 groups) resulted in neuronal impaired growth while dm2 model led to neuron hypertrophy in TG. Observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. In future, innovative strategies for modulation of CaMKIIα activity in specific subpopulations of neurons could be a novel approach in therapy of diabetic trigeminal neuropathy. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Calcium/Calmodulin Kinase II in the Pedunculopontine Tegmental Nucleus Modulates the Initiation and Maintenance of Wakefulness

    PubMed Central

    Datta, Subimal; O’Malley, Matthew W.; Patterson, Elissa H.

    2011-01-01

    The pedunculopontine tegmentum nucleus (PPT) is critically involved in the regulation of wakefulness (W) and rapid eye movement (REM) sleep, but our understanding of the mechanisms of this regulation remains incomplete. The present study was designed to determine the role of PPT intracellular calcium/calmodulin kinase (CaMKII) signaling in the regulation of W and sleep. To achieve this aim, three different concentrations (0.5, 1.0, and 2.0 nmol) of the CaMKII activation inhibitor, KN-93, were microinjected bilaterally (100 nl/site) into the PPT of freely moving rats, and the effects on W, slow-wave sleep (SWS), REM sleep, and levels of phosphorylated CaMKII (pCaMKII) expression in the PPT were quantified. These effects, which were concentration-dependent and affected wake–sleep variables for 3 h, resulted in decreased W, due to reductions in the number and duration of W episodes; increased SWS and REM sleep, due to increases in episode duration; and decreased levels of pCaMKII expression in the PPT. Regression analyses revealed that PPT levels of pCaMKII were positively related with the total percentage of time spentin W (R2 = 0.864; n = 28 rats; p < 0.001) and negatively related with the total percentage of time spentin sleep (R2 = 0.863; p < 0.001). These data provide the first direct evidence that activation of intracellular CaMKII signaling in the PPT promotes W and suppresses sleep. These findings are relevant for designing a drug that could treat excessive sleepiness by promoting alertness. PMID:22114270

  18. Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28)*♦

    PubMed Central

    Blackburn, R. Kevin; Monaghan, Jacqueline; Derbyshire, Paul; Menke, Frank L. H.; Zipfel, Cyril; Goshe, Michael B.; Zielinski, Raymond E.; Huber, Steven C.

    2017-01-01

    Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming. PMID:28154194

  19. Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways.

    PubMed

    Luo, Zhirong; Kohli, Meetu R; Yu, Qing; Kim, Syngcuk; Qu, Tiejun; He, Wen-xi

    2014-07-01

    Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. Biodentine is a bioactive and biocompatible material capable

  20. Hsp90 Enhances Degradation of Oxidized Calmodulin by the 20S Proteasome

    SciTech Connect

    Whittier, Jennifer E.; Xiong, Yijia; Rechsteiner, Martin C.; Squier, Thomas C.

    2004-10-29

    The 20S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress, and has been found in tight association with the molecular chaperone Hsp90. To elucidate the role of Hsp90 in promoting the degradation of oxidized calmodulin (CaMox), which accumulates in senescent brain during normal biological aging, we have purified the 20S proteasome free of Hsp90 from red blood cells and assessed its ability to recognize and degrade CaMox in the absence and presence of added Hsp90. The purified 20S proteasome does not degrade CaMox to any appreciable extent. However, following association with Hsp90, the 20S proteasome selectively degrades CaMox. This degradation is sensitive to both proteasome and Hsp90-specific inhibitors, and is further enhanced in the presence of 2 mM ATP. Irrespective of the presence of Hsp90 we find that unoxidized CaM is not significantly degraded. Furthermore, the ability of the proteasome to degrade commonly used fluorogenic peptides is not affected by Hsp90, indicating that there is no change in the accessibility of the catalytic core. Direct binding measurements demonstrate that Hsp90 selectively associates with CaMox; essentially no binding is observed between Hsp90 and unoxidized CaM. Since oxidation has previously been shown to induce both global conformational changes and a reduction in helical content of CaM, these results suggest that Hsp90 in association with the 20S proteasome selectively associates with partially unfolded proteins to promote their degradation by the proteasome.

  1. 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.

    PubMed

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-04-16

    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) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO-cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca(2+)/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO-cGMP and cAMP-PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors.

  2. Spontaneous Ca Waves in Ventricular Myocytes from Failing Hearts Depend on Ca2+-calmodulin-dependent Protein Kinase II

    PubMed Central

    Curran, Jerry; Brown, Kathy Hayes; Santiago, Demetrio J.; Pogwizd, Steve; Bers, Donald M.; Shannon, Thomas R.

    2010-01-01

    Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca]SRT) versus control. At similar [Ca]SRT (121 µM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 & 1.29±0.39 SCaWs cell−1, respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca]SRT. Inhibiting CaMKII reversed this effect (14%). At similar [Ca]SRT (71 µM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5±0.5; 1.6±0.7 vs. 0.36±0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis. PMID:20353795

  3. A Top-Down LC-FTICR MS-Based Strategy for Characterizing Oxidized Calmodulin in Activated Macrophages

    SciTech Connect

    Lourette, Natacha M.; Smallwood, Heather S.; Wu, Si; Robinson, Errol W.; Squier, Thomas C.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2010-06-01

    Liquid chromatography-mass spectrometry (LC-MS) based approach for monitoring time dependent changes in the degree of nitration and oxidation of intact calmodulin (CaM) has been used to resolve approximately 500 CaM oxiforms. Tentative identifications of posttranslational modifications (PTMs) such as oxidation or nitration have been assigned by combining tryptic peptide information (generated from bottom-up analyses) with online collision induced dissociation (CID) tandem mass spectrometry (MS/MS) at the intact protein level. The reduction in abundance and diversity of oxidatively modified CaM (i.e. nitrated tyrosines and oxidized methionines) induced by macrophage activation has been explored and semi-quantified for different oxidation degrees of CaM (i.e. no oxidation, moderate and high oxidation). This work demonstrates the power of top-down approach to identify hundreds of combinations of posttranslational modifications (PTMs) for single protein target such as CaM.

  4. Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

    PubMed Central

    Regulski, M; Tully, T

    1995-01-01

    Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates. Images Fig. 2 Fig. 3 PMID:7568075

  5. Current theories of neuronal information processing performed by Ca2+/calmodulin-dependent protein kinase II with support and insights from computer modelling and simulation.

    PubMed

    Coomber, C

    1998-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is concentrated in brain, and is particularly enriched in synaptic structures where it comprises 20-50% of all proteins. The abundant nature of CaMKII and its ability to phosphorylate a wide range of substrate proteins, including itself, earmarks it as a protein kinase that may have a vital role in neuronal information processing and memory. A computer model of CaMKII is investigated that incorporates recent findings about the geometrical arrangement of subunits, the mechanism of Ca(2+)-dependent subunit activation, and Ca(2+)-independent autophosphorylation. The model is framed as a system of nonlinear differential equations. It is demonstrated numerically that (1) CaMKII is tuned to be activated by stimulation protocols associated with the induction of long-term potentiation; (2) the observed slow dissociation of trapped Ca2+/calmodulin may require the autonomy site to be protected from dephosphorylation; and (3) Ca(2+)-independent kinase activity is expressed in a manner akin to a graded switch. The model validates current theories concerning how CaMKII may be a Ca2+ pulse frequency detector, a molecular switch, or a mediator of the threshold for long-term synaptic plasticity.

  6. Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells.

    PubMed

    Davletova, S; Mészáros, T; Miskolczi, P; Oberschall, A; Török, K; Magyar, Z; Dudits, D; Deák, M

    2001-02-01

    A calmodulin like domain protein kinase (CPK) homologue was identified in alfalfa and termed MsCPK3. The full-length sequence of cDNA encoded a 535 amino acid polypeptide with a molecular weight of 60.2 kDa. The deduced amino acid sequence showed all the conserved motifs that define other members of this kinase family, such as serine-threonine kinase domain, a junction region and four potential Ca2+ -binding EF sites. The recombinant MsCPK3 protein purified from E. coli was activated by Ca2+ and inhibited by calmodulin antagonist (W-7) in in vitro phosphorylation assays. The expression of MsCPK3 gene increased in the early phase of the 2,4-D induced alfalfa somatic embryogenesis. Heat shock also activated this gene while kinetin, ABA and NaCl treatment did not result in MsCPK3 mRNA accumulation. The data presented suggest that the new alfalfa CPK differs in stress responses from the previously described homologues and in its potential involvement in hormone and stress-activated reprogramming of developmental pathways during somatic embryogenesis.

  7. Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies.

    PubMed

    Naz, Huma; Shahbaaz, Mohd; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-06-01

    Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium-calmodulin dependent signal through a secondary messenger, Ca(2+), which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca(2+) concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0-11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0-4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV.

  8. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize.

    PubMed

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

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

  9. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Hidaka, H.

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

  10. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Hidaka, H.

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

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

    SciTech Connect

    Hansen, R.S.; Beavo, J.A.

    1986-11-05

    An anti-calmodulin monoclonal antibody having an absolute requirement for Ca/sup 2 +/ 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 Ca/sup 2 +//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.

  12. Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

    PubMed Central

    Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung

    2011-01-01

    Background Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue. Methods and Findings Single-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions. Conclusion The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in

  13. Regulation of the multifunctional Ca2+/calmodulin-dependent protein kinase II by the PP2C phosphatase PPM1F in fibroblasts.

    PubMed

    Harvey, Bohdan P; Banga, Satnam S; Ozer, Harvey L

    2004-06-04

    The regulation of the multifunctional calcium/calmodulin dependent protein kinase II (CaMKII) by serine/threonine protein phosphatases has been extensively studied in neuronal cells; however, this regulation has not been investigated previously in fibroblasts. We cloned a cDNA from SV40-transformed human fibroblasts that shares 80% homology to a rat calcium/calmodulin-dependent protein kinase phosphatase that encodes a PPM1F protein. By using extracts from transfected cells, PPM1F, but not a mutant (R326A) in the conserved catalytic domain, was found to dephosphorylate in vitro a peptide corresponding to the auto-inhibitory region of CaMKII. Further analyses demonstrated that PPM1F specifically dephosphorylates the phospho-Thr-286 in autophosphorylated CaMKII substrate and thus deactivates the CaMKII in vitro. Coimmunoprecipitation of CaMKII with PPM1F indicates that the two proteins can interact intracellularly. Binding of PPM1F to CaMKII involves multiple regions and is not dependent on intact phosphatase activity. Furthermore, overexpression of PPM1F in fibroblasts caused a reduction in the CaMKII-specific phosphorylation of the known substrate vimentin(Ser-82) following induction of the endogenous CaM kinase. These results identify PPM1F as a CaM kinase phosphatase within fibroblasts, although it may have additional functions intracellularly since it has been presented elsewhere as POPX2 and hFEM-2. We conclude that PPM1F, possibly together with the other previously described protein phosphatases PP1 and PP2A, can regulate the activity of CaMKII. Moreover, because PPM1F dephosphorylates the critical autophosphorylation site of CaMKII, we propose that this phosphatase plays a key role in the regulation of the kinase intracellularly.

  14. Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

    PubMed Central

    Wu, Pei-Rung; Kuo, Cheng-Chin; Yet, Shaw-Fang; Liou, Jun-Yang; Wu, Kenneth K.; Chen, Pei-Feng

    2012-01-01

    Background Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. Methodology/Principal Findings Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2–3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. Conclusions Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding. PMID:22768143

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

    PubMed Central

    Ataei, Negar; Sabzghabaee, Ali Mohammad; Movahedian, Ahmad

    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 considered and evaluated by the search engines PubMed, Cochrane Central Register of Controlled Trials and ScienceDirect Scopus between 1990 and February 2015. We did not carry out meta-analysis. Results: At the first search, it was fined 1015 articles which included “synaptic plasticity” OR “neuronal plasticity” OR “synaptic density” AND memory AND “molecular mechanism” AND “calcium/calmodulin-dependent protein kinase II” OR CaMKII as the keywords. A total of 335 articles were duplicates in the databases and eliminated. A total of 680 title articles were evaluated. Finally, 40 articles were selected as reference. 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. PMID:26445635

  16. Inhibitors of the Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

    SciTech Connect

    Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki; Sugiyama, Yasunori; Numano, Takamasa; Shigeri, Yasushi; Taniguchi, Takanobu; Kameshita, Isamu Ishida, Atsuhiko

    2007-11-23

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/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.

  17. Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae.

    PubMed

    Ohya, Y; Uno, I; Ishikawa, T; Anraku, Y

    1987-10-01

    Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.

  18. Dityrosine formation in calmodulin

    SciTech Connect

    Malencik, D.A.; Anderson, S.R.

    1987-02-10

    Ultraviolet (280-nm) irradiation of bovine brain calmodulin results in calcium-dependent changes in its fluorescence emission spectrum. These consist of a decline in the intrinsic tyrosine fluorescence of the protein and the appearance of a new emission maximum at 400 nm. Chromatography of irradiated calmodulin, using Ultrogel AcA 54 and phenyl-agarose columns, yields several distinctive fractions. One of these, representing 2.8% of the total recovered protein and 53% of the total fluorescence emission at 400 nm, was selected for detailed characterization. Analyses performed on acid hydrolysates reveal the presence of dityrosine, a derivative of tyrosine known for its fluorescence near 400 nm, at the level of 0.59-0.89 mol per 16,700 g of protein. Sodium dodecyl sulfate gel electrophoresis experiments demonstrate two components of apparent molecular weights 14,000 (80%) and 16,000 (20%). Observations on the effects of UV irradiation on the thrombic fragments of calmodulin and on related calcium binding proteins (rabbit skeletal muscle troponin C, bovine cardiac troponin C, and parvalbumin) support the interpretation that dityrosine formation in calmodulin results from the intramolecular cross-linking of Tyr-99 and Tyr-138. The dityrosine-containing photoproduct of calmodulin is unable to stimulate the p-nitrophenyl phosphatase activity of calcineurin under standard assay conditions. Smooth muscle myosin light chain kinase binds the derivative about 280-fold less effectively than it binds native calmodulin. Of several metal ions tested, only Cd/sup 2 +/ approaches Ca/sup 2 +/ in its ability to promote the appearance of the 400-nm emission band during UV irradiation of calmodulin. Mn/sup 2 +/ and Cu/sup 2 +/ appear to inhibit dityrosine formation.

  19. Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase

    PubMed Central

    Nagpal, Latika; Panda, Koustubh

    2015-01-01

    Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help

  20. AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca(2+)/calmodulin (CaM) dependence of Ca(2+)/CaM-dependent protein kinase kinase β.

    PubMed

    Nakanishi, Akihiro; Hatano, Naoya; Fujiwara, Yuya; Bin Shari, Arian; Takabatake, Shota; Akano, Hiroki; Kanayama, Naoki; Magari, Masaki; Nozaki, Naohito; Tokumitsu, Hiroshi

    2017-10-03

    The Ca(2+)/calmodulin-dependent protein kinase kinase β(CaMKKβ)/5'AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca(2+)-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca(2+) concentrations. Moreover, the Ca(2+)/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca(2+)/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr144 phosphorylation by activated AMPK converts CaMKKβ into a Ca(2+)/CaM-dependent enzyme, as shown by completely Ca(2+)/CaM-dependent CaMKK activity of a phosphomimetic Thr144Glu CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587) including the autoinhibitory region plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with antiphospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca(2+)-dependent AMPK activation by CaMKKβ. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  1. Behavioral modulation of neuronal calcium/calmodulin-dependent protein kinase II activity: differential effects on nicotine-induced spinal and supraspinal antinociception in mice.

    PubMed

    Damaj, M Imad

    2007-10-15

    Recent studies have implicated the involvement of Ca(2+)-dependent mechanisms, in particular calcium/calmodulin-dependent protein kinase II (CaM kinase II) in nicotine-induced antinociception using the tail-flick test. The spinal cord was suggested as a possible site of this involvement. The present study was undertaken to investigate the hypothesis that similar mechanisms exist for nicotine-induced antinociception in the hot-plate test, a response thought to be centrally mediated. In order to assess these mechanisms, i.c.v. administered CaM kinase II inhibitors were evaluated for their effects on antinociception produced by either i.c.v. or s.c. administration of nicotine in both tests. In addition, nicotine's analgesic effects were tested in mice lacking half of their CaM kinase II (CaM kinase II heterozygous) and compare it to their wild-type counterparts. Our results showed that although structurally unrelated CaM kinase II inhibitors blocked nicotine's effects in the tail-flick test in a dose-related manner, they failed to block the hot-plate responses. In addition, the antinociceptive effects of systemic nicotine in the tail-flick but not the hot-plate test were significantly reduced in CaM kinase II heterozygous mice. These observations indicate that in contrast to the tail-flick response, the mechanism of nicotine-induced antinociception in the hot-plate test is not mediated primarily via CaM kinase II-dependent mechanisms at the supraspinal level.

  2. Calcium supplementation during sepsis exacerbates organ failure and mortality via calcium/calmodulin-dependent protein kinase kinase (CaMKK) signaling

    PubMed Central

    Collage, Richard D.; Howell, Gina M.; Zhang, Xianghong; Stripay, Jennifer L.; Lee, Janet S.; Angus, Derek C.; Rosengart, Matthew R.

    2013-01-01

    Background Calcium plays an essential role in nearly all cellular processes. As such, cellular and systemic calcium concentrations are tightly regulated. During sepsis derangements in such tight regulation frequently occur, and treating hypocalcemia with parenteral calcium administration remains the current practice guideline. Objective We investigated whether calcium administration worsens mortality and organ dysfunction using an experimental murine model of sepsis and explored the mechanistic role of the family of calcium/calmodulin-dependent protein kinases in mediating these physiologic effects. To highlight the biological relevance of these observations, we conducted a translational study of the association between calcium administration, organ dysfunction and mortality among a cohort of critically ill septic ICU patients Design Prospective, randomized controlled experimental murine study. Observational clinical cohort analysis. Setting University research laboratory. Eight ICUs at a tertiary care center. Patients 870 septic ICU patients. Subjects C57BL/6 and CaMKK−/− mice. Interventions Mice underwent cecal ligation and puncture polymicrobial sepsis and were administered calcium chloride (0.25 or 0.25 mg/kg) or normal saline. Measurements and Main Results Administering calcium chloride to septic C57BL/6 mice heightened systemic inflammation and vascular leak, exacerbated hepatic and renal dysfunction, and increased mortality. These events were significantly attenuated in CaMKK−/− mice. In a risk–adjusted analysis of septic patients, calcium administration was associated with an increased risk of death, OR 1.92 (95% CI 1.00–3.68, p=0.049), a significant increase in the risk of renal dysfunction, OR 4.74 (95% CI 2.48–9.08, p<0.001), and a significant reduction in ventilator free days, mean decrease 3.29 days (0.50–6.08 days, p=0.02). Conclusions Derangements in calcium homeostasis occur during sepsis that are sensitive to calcium administration

  3. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  4. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  5. Dynamics of nitric oxide synthase-calmodulin interactions at physiological calcium concentrations.

    PubMed

    Piazza, Michael; Guillemette, J Guy; Dieckmann, Thorsten

    2015-03-24

    The intracellular Ca²⁺ concentration is an important regulator of many cellular functions. The small acidic protein calmodulin (CaM) serves as a Ca²⁺ sensor and control element for many enzymes. Nitric oxide synthase (NOS) is one of the proteins that is activated by CaM and plays a major role in a number of key physiological and pathological processes. Previous studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. We have analyzed the structure and dynamics of complexes formed by peptides based on inducible NOS (iNOS) and endothelial NOS (eNOS) with CaM at Ca²⁺ concentrations that mimic the physiological basal (17 and 100 nM) and elevated levels (225 nM) found in mammalian cells using fluorescence techniques and nuclear magnetic resonance spectroscopy. The results show the CaM-NOS complexes have similar structures at physiological and fully saturated Ca²⁺ levels; however, their dynamics are remarkably different. At 225 nM Ca²⁺, the CaM-NOS complexes show overall an increase in backbone dynamics, when compared to the dynamics of the complexes at saturating Ca²⁺ concentrations. Specifically, the N-lobe of CaM in the CaM-iNOS complex displays a lower internal mobility (higher S²) and higher exchange protection compared to those of the CaM-eNOS complex. In contrast, the C-lobe of CaM in the CaM-eNOS complex is less dynamic. These results illustrate that structures of CaM-NOS complexes determined at saturated Ca²⁺ concentrations cannot provide a complete picture because the differences in intramolecular dynamics become visible only at physiological Ca²⁺ levels.

  6. Phylogeny of Plant Calcium and Calmodulin-Dependent Protein Kinases (CCaMKs) and Functional Analyses of Tomato CCaMK in Disease Resistance

    PubMed Central

    Wang, Ji-Peng; Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

    2015-01-01

    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. PMID:26697034

  7. Innate immunity signaling: cytosolic Ca2+ elevation is linked to downstream nitric oxide generation through the action of calmodulin or a calmodulin-like protein.

    PubMed

    Ma, Wei; Smigel, Andries; Tsai, Yu-Chang; Braam, Janet; Berkowitz, Gerald A

    2008-10-01

    Ca(2+) rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca(2+)-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca(2+) rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca(2+)/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca(2+) signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca(2+) elevation, excluding the possibility of CaM acting upstream from Ca(2+). The CaM antagonist and Ca(2+) chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca(2+)/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca(2+) influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.

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

    PubMed

    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

  9. Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a G(o)/G(q) signaling network.

    PubMed Central

    Robatzek, M; Thomas, J H

    2000-01-01

    Caenorhabditis elegans locomotion is a complex behavior generated by a defined set of motor neurons and interneurons. Genetic analysis shows that UNC-43, the C. elegans Ca(2+)/calmodulin protein kinase II (CaMKII), controls locomotion rate. Elevated UNC-43 activity, from a gain-of-function mutation, causes severely lethargic locomotion, presumably by inappropriate phosphorylation of targets. In a genetic screen for suppressors of this phenotype, we identified multiple alleles of four genes in a G(o)/G(q) G-protein signaling network, which has been shown to regulate synaptic activity via diacylglycerol. Mutations in goa-1, dgk-1, eat-16, or eat-11 strongly or completely suppressed unc-43(gf) lethargy, but affected other mutants with reduced locomotion only weakly. We conclude that CaMKII and G(o)/G(q) pathways act in concert to regulate synaptic activity, perhaps through a direct interaction between CaMKII and G(o). PMID:11063685

  10. Bruton’s Tyrosine Kinase Mediates the Synergistic Signalling between TLR9 and the B Cell Receptor by Regulating Calcium and Calmodulin

    PubMed Central

    Kenny, Elaine F.; Quinn, Susan R.; Doyle, Sarah L.; Vink, Paul M.; van Eenennaam, Hans; O’Neill, Luke A. J.

    2013-01-01

    B cells signal through both the B cell receptor (BCR) which binds antigens and Toll-like receptors (TLRs) including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton’s tyrosine kinase (BTK) is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation. PMID:23967355

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

    SciTech Connect

    Riganti, Chiara

    2009-11-01

    Digoxin and ouabain are cardioactive glycosides, which inhibit the Na{sup +}/K{sup +}-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca{sup ++}]{sub 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{sup ++}]{sub i} and this event was dependent on the calcium influx via the Na{sup +}/Ca{sup ++} exchanger. The increased [Ca{sup ++}]{sub i} enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. 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{sup +}/Ca{sup ++} 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.

  12. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling

    PubMed Central

    Saddouk, Fatima Z.; Sun, Li-Yan; Liu, Yong Feng; Jiang, Miao; Singer, Diane V.; Backs, Johannes; Van Riper, Dee; Ginnan, Roman; Schwarz, John J.; Singer, Harold A.

    2016-01-01

    Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions.—Saddouk, F. Z., Sun, L.-Y., Liu, Y. F., Jiang, M., Singer, D. V., Backs, J., Van Riper, D., Ginnan, R., Schwarz, J. J., Singer, H. A. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling. PMID:26567004

  13. Medicago truncatula NIN Is Essential for Rhizobial-Independent Nodule Organogenesis Induced by Autoactive Calcium/Calmodulin-Dependent Protein Kinase1

    PubMed Central

    Marsh, John F.; Rakocevic, Alexandra; Mitra, Raka M.; Brocard, Lysiane; Sun, Jongho; Eschstruth, Alexis; Long, Sharon R.; Schultze, Michael; Ratet, Pascal; Oldroyd, Giles E.D.

    2007-01-01

    The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11∷GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules. PMID:17369436

  14. Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn*

    PubMed Central

    Ginnan, Roman; Zou, Xiaojing; Pfleiderer, Paul J.; Mercure, Melissa Z.; Barroso, Margarida; Singer, Harold A.

    2013-01-01

    In vascular smooth muscle (VSM) cells, Ca2+/calmodulin-dependent protein kinase IIδ2 (CaMKIIδ2) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ2 was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ2 and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ2 and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ2 and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ2 and Fyn, implicating CaMKIIδ2 as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ2 and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ2 and Fyn may coordinately regulate VSM cell motility. PMID:24003228

  15. Vascular smooth muscle cell motility is mediated by a physical and functional interaction of Ca2+/calmodulin-dependent protein kinase IIδ2 and Fyn.

    PubMed

    Ginnan, Roman; Zou, Xiaojing; Pfleiderer, Paul J; Mercure, Melissa Z; Barroso, Margarida; Singer, Harold A

    2013-10-11

    In vascular smooth muscle (VSM) cells, Ca(2+)/calmodulin-dependent protein kinase IIδ2 (CaMKIIδ2) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ2 was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ2 and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ2 and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ2 and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ2 and Fyn, implicating CaMKIIδ2 as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ2 and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ2 and Fyn may coordinately regulate VSM cell motility.

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

    PubMed

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

    2008-05-01

    Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide (H(2)O(2)), calcium (Ca(2+))-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, H(2)O(2), and CaCl(2) 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 Ca(2+) inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca(2+)-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 Ca(2+) in protoplasts of mesophyll cells and in the expression of calmodulin 1 (CaM1) 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 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca(2+) inhibitors and CaM antagonists. Our results suggest that Ca(2+)-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H(2)O(2)-induced antioxidant defense in leaves of maize plants.

  17. Characterization of a Central Ca2+/Calmodulin-dependent Protein Kinase IIα/β Binding Domain in Densin That Selectively Modulates Glutamate Receptor Subunit Phosphorylation*

    PubMed Central

    Jiao, Yuxia; Jalan-Sakrikar, Nidhi; Robison, A. J.; Baucum, Anthony J.; Bass, Martha A.; Colbran, Roger J.

    2011-01-01

    The densin C-terminal domain can target Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an “open” activated CaMKII conformation caused by Ca2+/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets. PMID:21610080

  18. Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+ /calmodulin-dependent protein kinase II

    PubMed Central

    Yao, Junlan; Davies, Lucinda A.; Howard, Jason D.; Adney, Scott K.; Welsby, Philip J.; Howell, Nancy; Carey, Robert M.; Colbran, Roger J.; Barrett, Paula Q.

    2006-01-01

    Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II–elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with α1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression. PMID:16917542

  19. Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    Yao, Junlan; Davies, Lucinda A; Howard, Jason D; Adney, Scott K; Welsby, Philip J; Howell, Nancy; Carey, Robert M; Colbran, Roger J; Barrett, Paula Q

    2006-09-01

    Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II-elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with alpha1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.

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

    PubMed Central

    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. Ca²⁺/calmodulin-dependent protein kinase II in the cockroach Periplaneta americana: identification of five isoforms and their tissues distribution.

    PubMed

    Taillebois, Emiliane; Heuland, Emilie; Bourdin, Céline M; Griveau, Audrey; Quinchard, Sophie; Tricoire-Leignel, Helene; Legros, Christian; Thany, Steeve H

    2013-07-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key kinase that transduces Ca²⁺ signals into downstream effects acting on a range of cellular processes in nervous system and muscular tissues. In insects, different CaMKII isoforms have been reported in Drosophila melanogaster, Apis florae, Bombus terrestris, and Bombus impatiens but little is known on the organization and tissue-specific expression of these isoforms with the exception of Drosophila. The present study reports the cloning of five CaMKII splice variants issued from a single gene and their tissue-specific expression in the cockroach Periplaneta americana. Each CaMKII isoform shared 82-90% identity with Drosophila CaMKII isoforms and accordingly were named PaCaMKII-A, PaCaMKII-B,PaCaMKII-C,PaCaMKII-D, and PaCaMKII-E. PaCaMKII-A and PaCaMKII-D isoforms are ubiquitously expressed in all tissues, but some such as PaCaMKII-B andPaCaMKII-C are preferentially expressed in the nerve cord and muscle. In addition, using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we found a tissue-specific expression of PaCaMKII-E in the dorsal unpaired median neurons. Alternative splicing of PaCaMKII transcripts is likely a common mechanism in insects to control the pattern of isoform expression in the different tissues.

  2. Signal transduction in lemon seedlings in the hypersensitive response against Alternaria alternata: participation of calmodulin, G-protein and protein kinases.

    PubMed

    Ortega, Ximena; Polanco, Rubén; Castañeda, Patricia; Perez, Luz M

    2002-01-01

    The development of an effective hypersensitive response (HR) in any plant system relies, not only in their gene composition and expression, but also on an effective and rapid signal transduction system. Lemon seedlings induce the phenylpropanoid pathway, which results in the de novo biosynthesis of the phytoalexin scoparone, as part of the hypersensitive response against Alternaria alternata. In order to elucidate some of the signaling elements that participate in the development of HR in lemon seedlings, we used several compounds that are known as activators or inhibitors of signal transduction elements in plants or in animal cells. Lemon seedlings treated either with cholera toxin or with phorbol 12-myristate 13-acetate (PMA), in the absence of A. alternata induced phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone, suggesting the participation of a G-protein and of a serine/threonine kinase, respectively, in signal transduction. The use of trifluoperazine (TFP), W-7, staurosporine, lavendustin A or 2,5-dihydroximethyl cinnamate (DHMC) prevented PAL induction as well as scoparone biosynthesis in response to the fungal inoculation, thus allowing us to infer the participation of Calmodulin (CaM), of serine/threonine and of tyrosine protein kinases (TPK) for signal transduction in Citrus limon in response to A. alternata.

  3. Dopamine D4 receptor transmission in the prefrontal cortex controls the salience of emotional memory via modulation of calcium calmodulin-dependent kinase II.

    PubMed

    Lauzon, Nicole M; Ahmad, Tasha; Laviolette, Steven R

    2012-11-01

    Dopamine (DA) signaling in the medial prefrontal cortex (mPFC) plays a critical role in the processing of emotional information and memory encoding. Activation of DA D4 receptors within the prelimbic (PLC) division of the mPFC bidirectionally modulates emotional memory by strongly potentiating the salience of normally nonsalient emotional memories but blocking the acquisition of suprathreshold emotionally salient fear memories. Previous in vitro studies have shown that activation of cortical DA D4 receptors can bidirectionally modulate levels of α-calcium calmodulin-dependent kinase II (α-CaMKII), a molecule essential for learning and memory. Using an olfactory fear conditioning procedure in rats combined with microinfusions into the mPFC, we examined the potential role of D4 receptor-mediated control of emotional memory salience through signaling via CaMKII, cAMP/protein kinase A (PKA), and protein phosphatase-1 (PP1) signaling. We report that CaMKII blockade prevents the ability of intra-mPFC DA D4 receptor activation to potentiate the salience of subthreshold fear memory. In contrast, blockade of either cAMP/PKA or PP1 signaling pathways rescued the blockade of suprathreshold fear memory via intra-mPFC D4 receptor activation. Our results demonstrate that modulation of emotional memory salience via intra-mPFC DA D4 receptor transmission depends upon downstream signaling via CaMKII, cAMP/PKA, and PP1 substrates.

  4. Intra-nucleus accumbens administration of the calcium/calmodulin-dependent protein kinase II inhibitor AIP induced antinociception in rats with mononeuropathy.

    PubMed

    Bian, Hui; Yu, Long-Chuan

    2015-07-10

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine- dependent protein kinase, which has been implicated in pain modulation at different levels of the central nervous system. The present study was performed in rats with mononeuropathy induced by left common sciatic nerve ligation. Unilateral sciatic nerve loose ligation produced decreases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation. Intra-nucleus accumbens (NAc) injection of 3 μg, 6 μg and 12 μg of myristoylated autocamtide-2-inhibitory peptide (AIP), the CaMKII inhibitor, dose-dependently increased the HWL to noxious thermal and mechanical stimulation in rats with mononeuropathy. Furthermore, intra-NAc administration of morphine, the HWL to noxious thermal and mechanical stimulation increased markedly, and there were no significant differences between morphine group and AIP group. Taken together, the results showed that intra-NAc injection of AIP induced significant antinociceptive effects in rats with mononeuropathy, indicating that CaMKII may play an important role in the transmission and/or modulation of nociceptive information in the NAc in rats with mononeuropathy.

  5. Developmental differences in posttranslational calmodulin methylation in pea plants

    SciTech Connect

    Oh, Sukheung; Roberts, D.M. )

    1990-05-01

    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 {sup 3}H-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.

  6. Regional distribution and subcellular associations of Type II calcium and calmodulin-dependent protein kinase in rat brain

    SciTech Connect

    Erondu, N.E.

    1986-01-01

    Four monoclonal antibodies generated against the Type II CaM kinase have been characterized. Two of these antibodies were used to confirm that both alpha and beta subunits were part of the holoenzyme complex. I also developed liquid phase and solid phase radioimmunoassays for the kinase. With the solid phase radioimmunoassay, the distribution of the kinase in rat brain was examined. This study revealed that the concentration of the kinase varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of total hippocampal protein, 1.3% of cortical protein and 0.7% of striatal protein. It is less concentrated in lower brain regions ranging from 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla.

  7. Involvement of calcium and calmodulin in oxidative and temperature stress of Amaranthus lividus L. during early germination.

    PubMed

    Bhattacharjee, Soumen

    2009-07-01

    Both heat and chilling caused reduction in membrane protein thiol level and increased accumulation of thiobarbituric acid reactive substances in 72 hr old germinating tissues (indicators of oxidative stress) and reduced germination and early growth performances. Calcium chelator EGTA [Ethylene glycol-bis (2-aminoethylether)-N, N,N',N, tetra acetic acid] calcium channel blocker LaCI3 (Lanthanum chloride) and calmodulin inhibitor TFP (trifluroperazine) aggravated these effects of heat and chilling and added calcium reversed them. Imposition of heat and chilling stress during early germination also causes accumulation of reactive oxygen species (ROS) like 02(-) and H2O2. Calcium treatment significantly reduced the accumulation of both the toxic ROS, while EGTA, LaCl3 and TFP treatment enhanced the accumulation. Activities of antioxidative enzymes catalase (CAT), ascorbate peroxidase (APOX) and glutathione reductase (GR) and total thiol content decreased significantly under both heat and chilling stress in germinating Amaranthus seedlings. Seedlings raised with Ca2+ treatment under heat and chilling stress exhibit higher activities of CAT7 GR and APOX and total thiol level than the untreated plants. EGTA, LaCl3 and TFP treatment, on the other hand significantly reduce the activities of all anti-oxidative enzymes and total thiol level. The work clearly supports the view that Ca2+-signalling pathway plays significant role in limiting heat and chilling induced oxidative stress by upregulating antioxidative defense during recovery phase of post-germination event in Amaranthus lividus.

  8. L-type calcium channels and calcium/calmodulin-dependent kinase II differentially mediate behaviors associated with nicotine withdrawal in mice.

    PubMed

    Jackson, K J; Damaj, M I

    2009-07-01

    Smoking is a widespread health problem. Because the nicotine withdrawal syndrome is a major contributor to continued smoking and relapse, it is important to understand the molecular and behavioral mechanisms of nicotine withdrawal to generate more effective smoking cessation therapies. Studies suggest a role for calcium-dependent mechanisms, such as L-type calcium channels and calcium/calmodulin-dependent protein kinase II (CaMKII), in the effects of nicotine dependence; however, the role of these mechanisms in nicotine-mediated behaviors is unclear. Thus, the goal of this study was to elucidate the role of L-type calcium channels and CaMKII in nicotine withdrawal behaviors. Using both pharmacological and genetic methods, our results show that L-type calcium channels are involved in physical, but not affective, nicotine withdrawal behaviors. Although our data do provide evidence of a role for CaMKII in nicotine withdrawal behaviors, our pharmacological and genetic assessments yielded different results concerning the specific role of the kinase. Pharmacological data suggest that CaMKII is involved in somatic signs and affective nicotine withdrawal, and activity level is decreased after nicotine withdrawal, whereas the genetic assessments yielded results suggesting that CaMKII is involved only in the anxiety-related response, yet the kinase activity may be increased after nicotine withdrawal; thus, future studies are necessary to clarify the precise behavioral specifics of the relevance of CaMKII in nicotine withdrawal behaviors. Overall, our data show that L-type calcium channels and CaMKII are relevant in nicotine withdrawal and differentially mediate nicotine withdrawal behaviors.

  9. Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin.

    PubMed

    Robison, A J; Bartlett, Ryan K; Bass, Martha A; Colbran, Roger J

    2005-11-25

    Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.

  10. Gender-Specific Potential Inhibitory Role of Ca2+/Calmodulin Dependent Protein Kinase Phosphatase (CaMKP) in Pressure-Overloaded Mouse Heart

    PubMed Central

    Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

    2014-01-01

    Background Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Methodology and Principal Findings Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Conclusions/Significance Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts

  11. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  12. Effect of exercise on synaptophysin and calcium/calmodulin-dependent protein kinase levels in prefrontal cortex and hippocampus of a rat model of developmental stress

    PubMed Central

    Hescham, Sarah; Grace, Laurian; Kellaway, Lauriston A; Bugarith, Kishor; Russell, Vivienne A

    2010-01-01

    Stress affects the brain differently depending on the timing, duration and intensity of the stressor. Separation from the dam for 3 hours per day is a potent stressor for rat pups which causes activation of the hypothalamic-pituitary-adrenal (HPA) axis, evidenced by increased plasma levels of adrenocorticotropin (ACTH) and glucocorticoids. Behaviourally, animals display anxiety-like behaviour while structurally, changes occur in neuronal dendrites and spines in the hippocampus and prefrontal regions involved in emotion and behaviour control. The aim of the present study was to determine whether maternal separation alters expression of synaptic markers, synaptophysin and calcium/calmodulin-dependent protein kinase II, CaMKII, in rat hippocampus and prefrontal cortex. A second aim was to determine whether voluntary exercise had a beneficial effect on the expression of these proteins in rat brain. Maternal separation occurred from postnatal day 2 (P2) to P14 for 3 hours per day. Exercised rats were housed in cages with attached running wheels from P29 to P49. At P65, the prefrontal cortex and hippocampus were removed for protein quantification. Maternal separation did not have any effect while exercise increased synaptophysin and CaMKII in the ventral hippocampus but not in the dorsal hippocampus or prefrontal cortex. Since the ventral hippocampus is associated with anxiety-related behaviour, these findings are consistent with the fact that voluntary exercise increases anxiety-like behaviour and improves learning and memory. PMID:19821017

  13. Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

    PubMed Central

    Lladó, Anna; Timpson, Paul; Vilà de Muga, Sandra; Moretó, Jemina; Pol, Albert; Grewal, Thomas; Daly, Roger J.

    2008-01-01

    The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. PMID:17959830

  14. Ca2+/calmodulin protein kinase II and memory: learning-related changes in a localized region of the domestic chick brain.

    PubMed

    Solomonia, Revaz O; Kotorashvili, Adam; Kiguradze, Tamar; McCabe, Brian J; Horn, Gabriel

    2005-12-01

    The role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the recognition memory of visual imprinting was investigated. Domestic chicks were exposed to a training stimulus and learning strength measured. Trained chicks, together with untrained chicks, were killed either 1 h or 24 h after training. The intermediate and medial hyperstriatum ventrale/mesopallium (IMHV/IMM), a forebrain memory storage site, was removed together with a control brain region, the posterior pole of the neostriatum/nidopallium (PPN). Amounts of membrane total alphaCaMKII (tCaMKII) and Thr286-autophosphorylated alphaCaMKII (apCAMKII) were measured. For the IMHV/IMM 1 h group, apCaMKII amount and apCAMKII/tCaMKII increased as chicks learned. The magnitude of the molecular changes were positively correlated with learning strength. No learning-related effects were observed in PPN, or in either region at 24 h. These results suggest that CaMKII is involved in the formation of memory but not in its maintenance.

  15. The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

    PubMed Central

    Li, Changlin; Cai, Xiangyu; Sun, Haili; Bai, Ting; Zheng, Xilong; Zhou, Xing Wang; Chen, Xiongwen; Gill, Donald L.; Li, Jing; Tang, Xiang D.

    2011-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are 3 δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target. PMID:21554860

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

    PubMed

    Haolong, Cong; Du, Ning; Hongchao, Tian; Yang, Yang; Wei, Zhang; Hua, Zhang; Wenliang, Zhang; Lei, Song; Po, Tien

    2013-01-01

    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.

  17. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling.

    PubMed

    Saddouk, Fatima Z; Sun, Li-Yan; Liu, Yong Feng; Jiang, Miao; Singer, Diane V; Backs, Johannes; Van Riper, Dee; Ginnan, Roman; Schwarz, John J; Singer, Harold A

    2016-03-01

    Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions. © FASEB.

  18. Curcumin Inhibits Neuronal Loss in the Retina and Elevates Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Diabetic Rats

    PubMed Central

    Wang, Peipei; Zhu, Yanxia; Chen, Zhen; Shi, Tianyan; Lei, Wensheng

    2015-01-01

    Abstract Purpose: To determine whether curcumin offers neuroprotection to minimize the apoptosis of neural cells in the retina of diabetic rats. Methods: Streptozotocin (STZ)-induced diabetic rats and control rats were used in this study. A subgroup of STZ-induced diabetic rats were treated with curcumin for 12 weeks. Retinal histology, apoptosis of neural cells in the retina, electroretinograms, and retinal glutamate content were evaluated after 12 weeks. Retinal levels of Ca2+/calmodulin-dependent protein kinase II (CaMKII), phospho-CaMKII (p-CaMKII), and cleaved caspase-3 were determined by Western blot analysis. Results: The amplitudes a-wave, b-wave, and oscillatory potential were reduced by diabetes, but curcumin treatment suppressed this reduction of amplitudes. Curcumin also prevented cell loss from the outer nuclear, inner nuclear, and ganglion cell layers. Apoptosis of retinal neurons was detected in diabetic rats. The concentration of glutamate in the retina was higher in diabetic rats, but was significantly reduced in the curcumin-treated group. Furthermore, p-CaMKII and cleaved caspase-3 expression were upregulated in the diabetic retina, but reduced in curcumin-treated rats. Conclusions: Curcumin attenuated diabetes-induced apoptosis in retinal neurons by reducing the glutamate level and downregulating CaMKII. Thus, curcumin might be used to prevent neuronal damage in the retina of patients with diabetes mellitus. PMID:26207889

  19. Wenxin-Keli Regulates the Calcium/Calmodulin-Dependent Protein Kinase II Signal Transduction Pathway and Inhibits Cardiac Arrhythmia in Rats with Myocardial Infarction

    PubMed Central

    Xing, Yanwei; Gao, Yonghong; Chen, Jianxin; Zhu, Haiyan; Wu, Aiming; Yang, Qing; Teng, Fei; Zhang, Dong-mei; Xing, Yanhui; Gao, Kuo; He, Qingyong; Zhang, Zhenpeng; Wang, Jie; Shang, Hongcai

    2013-01-01

    Wenxin-Keli (WXKL) is a Chinese herbal compound reported to be of benefit in the treatment of cardiac arrhythmia, cardiac inflammation, and heart failure. Amiodarone is a noncompetitive inhibitor of the α- and β-adrenergic receptors and prevents calcium influx in the slow-response cells of the sinoatrial and atrioventricular nodes. Overexpression of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in transgenic mice results in heart failure and arrhythmias. We hypothesised that administration of WXKL and amiodarone can reduce the incidence of arrhythmias by regulating CaMKII signal transduction. A total of 100 healthy Sprague Dawley rats were used in the study. The rats were randomly divided into four groups (a sham group, a myocardial infarction (MI) group, a WXKL-treated group, and an amiodarone-treated group). A myocardial infarction model was established in these rats by ligating the left anterior descending coronary artery for 4 weeks. Western blotting was used to assess CaMKII, p-CaMKII (Thr-286), PLB, p-PLB (Thr-17), RYR2, and FK binding protein 12.6 (FKBP12.6) levels. The Ca2+ content in the sarcoplasmic reticulum (SR) and the calcium transient amplitude were studied by confocal imaging using the fluorescent indicator Fura-4. In conclusion, WXKL may inhibit heart failure and cardiac arrhythmias by regulating the CaMKII signal transduction pathway similar to amiodarone. PMID:23781262

  20. Wenxin-Keli Regulates the Calcium/Calmodulin-Dependent Protein Kinase II Signal Transduction Pathway and Inhibits Cardiac Arrhythmia in Rats with Myocardial Infarction.

    PubMed

    Xing, Yanwei; Gao, Yonghong; Chen, Jianxin; Zhu, Haiyan; Wu, Aiming; Yang, Qing; Teng, Fei; Zhang, Dong-Mei; Xing, Yanhui; Gao, Kuo; He, Qingyong; Zhang, Zhenpeng; Wang, Jie; Shang, Hongcai

    2013-01-01

    Wenxin-Keli (WXKL) is a Chinese herbal compound reported to be of benefit in the treatment of cardiac arrhythmia, cardiac inflammation, and heart failure. Amiodarone is a noncompetitive inhibitor of the α - and β -adrenergic receptors and prevents calcium influx in the slow-response cells of the sinoatrial and atrioventricular nodes. Overexpression of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in transgenic mice results in heart failure and arrhythmias. We hypothesised that administration of WXKL and amiodarone can reduce the incidence of arrhythmias by regulating CaMKII signal transduction. A total of 100 healthy Sprague Dawley rats were used in the study. The rats were randomly divided into four groups (a sham group, a myocardial infarction (MI) group, a WXKL-treated group, and an amiodarone-treated group). A myocardial infarction model was established in these rats by ligating the left anterior descending coronary artery for 4 weeks. Western blotting was used to assess CaMKII, p-CaMKII (Thr-286), PLB, p-PLB (Thr-17), RYR2, and FK binding protein 12.6 (FKBP12.6) levels. The Ca(2+) content in the sarcoplasmic reticulum (SR) and the calcium transient amplitude were studied by confocal imaging using the fluorescent indicator Fura-4. In conclusion, WXKL may inhibit heart failure and cardiac arrhythmias by regulating the CaMKII signal transduction pathway similar to amiodarone.

  1. Calcium/calmodulin-dependent protein kinase IIbeta isoform is expressed in motor neurons during axon outgrowth and is part of slow axonal transport.

    PubMed

    Lund, Linda M; McQuarrie, Irvine G

    2002-03-15

    Previously, we identified calcium/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) mRNA in spinal motor neurons with 372 bp inserted in what corresponds to the "association" domain of the protein. This was interesting because known additions and deletions to CaMKIIbeta mRNA are usually less than 100 bp in size and found in the "variable" region. Changes in the association domain of CaMKIIbeta could influence substrate specificity, activity or intracellular targeting. We show that three variations of this insert are found in CNS neurons or sciatic motor neurons of Sprague-Dawley rats. We used PCR and nucleic acid sequencing to identify inserts of 114, 243, or 372 bases. We also show that addition of the 372 bases is associated with outgrowth of the axon (the standard CaMKIIbeta downregulates when axon outgrowth occurs). Radiolabeling, immunoblots, and 2D PAGE identified this larger CaMKIIbeta as part of the group of soluble proteins moving at the slowest rate of axonal transport (SCa) in sciatic motor neurons (similar1 mm/day). This group is composed mainly of structural proteins (e.g., tubulin) used to assemble the cytoskeleton of regrowing axons.

  2. Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy.

    PubMed

    Pfisterer, Simon G; Mauthe, Mario; Codogno, Patrice; Proikas-Cezanne, Tassula

    2011-12-01

    Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca(2+)-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5-50 μM), sorafenib (1-40 μM), staurosporine (20-500 nM), or thapsigargin (20-500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca(2+) chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10-30 μg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1-10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca(2+) mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α(1)/α(2)-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down

  3. Reactive sulfur species inactivate Ca(2+)/calmodulin-dependent protein kinase IV via S-polysulfidation of its active-site cysteine residue.

    PubMed

    Takata, Tsuyoshi; Ihara, Hideshi; Hatano, Naoya; Tsuchiya, Yukihiro; Akaike, Takaaki; Watanabe, Yasuo

    2017-07-17

    Reactive sulfur species (RSS) modulate protein functions via S-polysulfidation of reactive Cys residues. Here, we report that Ca(2+)/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr(196) by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na2S n (n = 2-4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr(196) and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na2S n -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys(198) in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr(196) and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr(196) was decreased upon treatment with either Na2S4 or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr(196) of endogenous CaMKIV was also inhibited by treatment either with Na2S4 or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S-polysulfidation of its Cys(198) during the response to ER stress. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  4. Phosphorylation of calcium/calmodulin-dependent protein kinase II in the rat dorsal medial prefrontal cortex is associated with alcohol-induced cognitive inflexibility.

    PubMed

    Natividad, Luis A; Steinman, Michael Q; Laredo, Sarah A; Irimia, Cristina; Polis, Ilham Y; Lintz, Robert; Buczynski, Matthew W; Martin-Fardon, Rémi; Roberto, Marisa; Parsons, Loren H

    2017-09-22

    Repeated cycles of alcohol [ethanol (EtOH)] intoxication and withdrawal dysregulate excitatory glutamatergic systems in the brain and induce neuroadaptations in the medial prefrontal cortex (mPFC) that contribute to cognitive dysfunction. The mPFC is composed of subdivisions that are functionally distinct, with dorsal regions facilitating drug-cue associations and ventral regions modulating new learning in the absence of drug. A key modulator of glutamatergic activity is the holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) that phosphorylates ionotropic glutamate receptors. Here, we examined the hypothesis that abstinence from chronic intermittent EtOH (CIE) exposure dysregulates CaMKII activity in the mPFC to impair cognitive flexibility. We used an operant model of strategy set shifting in male Long-Evans rats demonstrating reduced susceptibility to trial omissions during performance in a visual cue-guided task versus albino strains. Relative to naïve controls, rats experiencing approximately 10 days of abstinence from CIE vapor exposure demonstrated impaired performance during a procedural shift from visual cue to spatial location discrimination. Phosphorylation of CaMKII subtype α was upregulated in the dorsal, but not ventral mPFC of CIE-exposed rats, and was positively correlated with perseverative-like responding during the set shift. The findings suggest that abstinence from CIE exposure induces an undercurrent of kinase activity (e.g. CaMKII), which may promote aberrant glutamatergic responses in select regions of the mPFC. Given the role of the mPFC in modulating executive control of behavior, we propose that increased CaMKII subtype α activity reflects a dysregulated 'top-down' circuit that interferes with adaptive behavioral performance under changing environmental demands. © 2017 Society for the Study of Addiction.

  5. Cyclin-dependent kinase 5 is a calmodulin-binding protein that associates with puromycin-sensitive aminopeptidase in the nucleus of Dictyostelium.

    PubMed

    Huber, Robert J; Catalano, Andrew; O'Day, Danton H

    2013-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 ((132)LLINRKGELKLADFGLARAFGIP(154)) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP.

  6. Human calcium/calmodulin-dependent serine protein kinase regulates the expression of p21 via the E2A transcription factor.

    PubMed

    Sun, Rongju; Su, Yongyue; Zhao, Xiaodong; Qi, Jie; Luo, Xiaofeng; Yang, Zongcheng; Yao, Yongming; Luo, Xiangdong; Xia, Zhaofan

    2009-04-15

    CASK (calcium/calmodulin-dependent serine protein kinase) is a kind of scaffolding protein that recruits or organizes other proteins at the plasma membrane to co-ordinate signal transduction pathways within the cytoplasm and nucleus. We have previously found that hCASK (human CASK) binds Id1 (inhibitor of DNA binding 1) through hCASK's GUK (guanylate kinase) domain and inhibits cell growth, probably via interactions with Id1. Overexpression of hCASK resulted in a reduced rate of cell growth, although inhibition of CASK via RNAi (RNA interference) promoted cell proliferation in ECV304 cells. This study revealed that hCASK regulates the protein and mRNA level of p21(wafi/cip1) (referred to throughout as p21), and activated the expression of p21 in a time-dependent manner. Two E-boxes in the proximal region at the TSS (transcription start site) play key roles in regulating hCASK-mediated p21 expression. We suggest that E2A (E12 and E47), a representative of the E proteins that binds the E-box elements, is a participant in the mediation of p21 expression by hCASK. The results of the present study suggest that hCASK regulation of cell growth might involve p21 expression, and that the bHLH (basic helix-loop-helix) transcription factor E2A probably participates in hCASK regulation of p21 expression. From these findings, we propose a novel proliferation signalling pathway mediated by hCASK.

  7. Aberrant calcium/calmodulin-dependent protein kinase II (CaMKII) activity is associated with abnormal dendritic spine morphology in the ATRX mutant mouse brain.

    PubMed

    Shioda, Norifumi; Beppu, Hideyuki; Fukuda, Takaichi; Li, En; Kitajima, Isao; Fukunaga, Kohji

    2011-01-05

    In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the sucrose-nonfermenting 2 family, cause several mental retardation disorders, including α-thalassemia X-linked mental retardation syndrome. We generated ATRX mutant mice lacking exon 2 (ATRX(ΔE2) mice), a mutation that mimics exon 2 mutations seen in human patients and associated with milder forms of retardation. ATRX(ΔE2) mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). Consistent with other mouse models of mental retardation, ATRX(ΔE2) mice exhibited longer and thinner dendritic spines compared with wild-type mice without changes in spine number. Interestingly, aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRX(ΔE2) mice. Increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-guanine nucleotide exchange factors (GEFs) T-cell lymphoma invasion and metastasis 1 (Tiam1) and kalirin-7, known substrates of CaMKII. We confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRX(ΔE2) mice. In cultured cortical neurons, PP1 inhibition by okadaic acid increased CaMKII-dependent Tiam1 and kalirin-7 phosphorylation. Together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes plus elevated Rac1-GEF/PAK signaling seen in ATRX(ΔE2) mice may contribute to mental retardation syndromes seen in human patients.

  8. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo.

    PubMed

    Jackson, K J; Walters, C L; Damaj, M I

    2009-08-01

    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

  9. 5-Hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms.

    PubMed

    Berg, K A; Clarke, W P; Chen, Y; Ebersole, B J; McKay, R D; Maayani, S

    1994-05-01

    The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 microM) amplified the amount of cAMP formed in response to 5'-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5'-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (alpha, delta, and epsilon), only the calcium-independent isoform PKC-epsilon remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.

  10. Modulation of calcium-mediated inactivation of ionic currents by Ca2+/calmodulin-dependent protein kinase II.

    PubMed Central

    Sakakibara, M; Alkon, D L; DeLorenzo, R; Goldenring, J R; Neary, J T; Heldman, E

    1986-01-01

    Iontophoretic injection of Ca2+ causes reduction of I0A (an early rapidly activating and inactivating K+ current) and I0C (a late Ca2+-dependent K+ current) measured across the isolated type B soma membrane (Alkon et al., 1984, 1985; Alkon and Sakakibara, 1984, 1985). Similarly, voltage-clamp conditions which cause elevation of [Ca2+]i are followed by reduction of I0A and I0C lasting 1-3 min. Iontophoretic injection of highly purified Ca2+/CaM-dependent protein kinase II (CaM kinase II) isolated from brain tissue (Goldenring et al., 1983) enhanced and prolonged this Ca2+-mediated reduction of I0A and I0C. ICa2+, a voltage-dependent Ca2+ current, also showed some persistent reduction under these conditions. Iontophoretic injection of heat-inactivated enzyme had no effect. Agents that inhibit or block Ca2+/CaM-dependent phosphorylation produced increased I0A and I0C amplitudes and prevented the effects of CaM kinase II injection. The results reported here and in other studies implicate Ca2+-stimulated phosphorylation in the regulation of type B soma ionic currents. PMID:2427133

  11. Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle.

    PubMed

    Damiani, E; Sacchetto, R; Margreth, A

    2000-12-09

    Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.

  12. Oxidatively modified calmodulin binds to the plasma membrane Ca-ATPase in a nonproductive and conformationally disordered complex.

    PubMed Central

    Gao, J; Yao, Y; Squier, T C

    2001-01-01

    Oxidation of either Met(145) or Met(146) in wheat germ calmodulin (CaM) to methionine sulfoxide prevents the CaM-dependent activation of the plasma membrane (PM) Ca-ATPase (D. Yin, K. Kuczera, and T. C. Squier, 2000, Chem. Res. Toxicol. 13:103-110). To investigate the structural basis for the inhibition of the PM-Ca-ATPase by oxidized CaM (CaM(ox)), we have used circular dichroism (CD) and fluorescence spectroscopy to resolve conformational differences within the complex between CaM and the PM-Ca-ATPase. The similar excited-state lifetime and solvent accessibility of the fluorophore N-1-pyrenyl-maleimide covalently bound to Cys(26) in unoxidized CaM and CaM(ox) indicates that the globular domains within CaM(ox) assume a native-like structure following association with the PM-Ca-ATPase. However, in comparison with oxidized CaM there are increases in the 1) molar ellipticity in the CD spectrum and 2) conformational heterogeneity between the opposing globular domains for CaM(ox) bound to the CaM-binding sequence of the PM-Ca-ATPase. Furthermore, CaM(ox) binds to the PM-Ca-ATPase with high affinity at a distinct, but overlapping, site to that normally occupied by unoxidized CaM. These results suggest that alterations in binding interactions between CaM(ox) and the PM-Ca-ATPase block important structural transitions within the CaM-binding sequence of the PM-Ca-ATPase that are normally associated with enzyme activation. PMID:11259292

  13. Ca2+/Calmodulin-dependent Protein Kinase II Inhibitors Disrupt AKAP79-dependent PKC Signaling to GluA1 AMPA Receptors*

    PubMed Central

    Brooks, Ian M.; Tavalin, Steven J.

    2011-01-01

    GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31–52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31–52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31–52) in the absence (apoCaM) but not the presence of Ca2+. In contrast, apoCaM association to AKAP79(31–52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31–52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII. PMID:21156788

  14. A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II.

    PubMed

    Welsby, Philip J; Wang, Hongge; Wolfe, Joshua T; Colbran, Roger J; Johnson, Michael L; Barrett, Paula Q

    2003-11-05

    Low-voltage-activated (LVA) Ca2+ channels are widely distributed throughout the CNS and are important determinants of neuronal excitability, initiating dendritic and somatic Ca2+ spikes that trigger and shape the pattern of action potential firing. Here, we define a molecular mechanism underlying the dynamic regulation of alpha1H channels (Cav3.2), by Ca2+/CaM-dependent protein kinase II (CaMKII). We show that channel regulation is selective for the LVA alpha1H Ca2+ channel subtype, depends on determinants in the alpha1H II-III intracellular loop, and requires the phosphorylation of a serine residue absent from unregulated alpha1G (Cav3.1) channels. These studies identify the alpha1H channel as a new substrate for CaMKII and provide the first molecular mechanism for the direct regulation of T-type Ca2+ channels by a protein kinase. Our data suggest a novel mechanism for modulating the integrative properties of neurons.

  15. Rice SPK, a Calmodulin-Like Domain Protein Kinase, Is Required for Storage Product Accumulation during Seed Development

    PubMed Central

    Asano, Takayuki; Kunieda, Noriko; Omura, Yuhi; Ibe, Hirokazu; Kawasaki, Tsutomu; Takano, Makoto; Sato, Miho; Furuhashi, Hideyuki; Mujin, Toshiyuki; Takaiwa, Fumio; Wu, Chuan-yin; Tada, Yuichi; Satozawa, Tomomi; Sakamoto, Masahiro; Shimada, Hiroaki

    2002-01-01

    Suc, an end product of photosynthesis, is metabolized by Suc synthase in sink organs as an initial step in the biosynthesis of storage products. Suc synthase activity is known to be regulated by reversible phosphorylation, but the details of this process are unclear at present. Rice SPK, a calcium-dependent protein kinase, is expressed uniquely in the endosperm of immature seed, and its involvement in the biosynthetic pathways of storage products was suggested. Antisense SPK transformants lacked the ability to accumulate storage products such as starch, but produced watery seed with a large amount of Suc instead, as the result of an inhibition of Suc degradation. Analysis of in vitro phosphorylation indicated that SPK phosphorylated specifically a Ser residue in Suc synthase that has been shown to be important for its activity in the degradation of Suc. This finding suggests that SPK is involved in the activation of Suc synthase. It appears that SPK is a Suc synthase kinase that may be important for supplying substrates for the biosynthesis of storage products. PMID:11910009

  16. Pulsed Electron Paramagnetic Resonance Study of Domain Docking in Neuronal Nitric Oxide Synthase: The Calmodulin and Output State Perspective

    PubMed Central

    2015-01-01

    The binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) enables formation of the output state of nNOS for nitric oxide production. Essential to NOS function is the geometry and dynamics of CaM docking to the NOS oxygenase domain, but little is known about these details. In the present work, the domain docking in a CaM-bound oxygenase/FMN (oxyFMN) construct of nNOS was investigated using the relaxation-induced dipolar modulation enhancement (RIDME) technique, which is a pulsed electron paramagnetic resonance technique sensitive to the magnetic dipole interaction between the electron spins. A cysteine was introduced at position 110 of CaM, after which a nitroxide spin label was attached at the position. The RIDME study of the magnetic dipole interaction between the spin label and the ferric heme centers in the oxygenase domain of nNOS revealed that, with increasing [Ca2+], the concentration of nNOS·CaM complexes increases and reaches a maximum at [Ca2+]/[CaM] ≥ 4. The RIDME kinetics of CaM-bound nNOS represented monotonous decays without well-defined oscillations. The analysis of these kinetics based on the structural models for the open and docked states has shown that only about 15 ± 3% of the CaM-bound nNOS is in the docked state at any given time, while the remaining 85 ± 3% of the protein is in the open conformations characterized by a wide distribution of distances between the bound CaM and the oxygenase domain. The results of this investigation are consistent with a model that the Ca2+–CaM interaction causes CaM docking with the oxygenase domain. The low population of the docked state indicates that the CaM-controlled docking between the FMN and heme domains is highly dynamic. PMID:25046446

  17. Jieduquyuziyin prescription suppresses IL-17 production and Th17 activity in MRL/lpr mice by inhibiting expression of Ca(2+)/calmodulin-dependent protein kinase-4.

    PubMed

    Shui, Bingjie; Xia, Weibiao; Wen, Chengping; Ding, Xinghong

    2015-07-01

    Jieduquyuziyin prescription (JP) is a traditional Chinese medicinal (TCM) formula which has been demonstrated to be effective for systemic lupus erythematosus (SLE) treatment as an approved hospital prescription for many years in China. But its mechanism of action in combating this disease is largely unknown. Our previous studies showed that JP can slow disease progression without producing significant toxic side effects. We treated MRL/lpr mice with JP to ascertain if JP could improve SLE by the suppression of Ca(2+)/calmodulin-dependent protein kinase-4 (CaMK4) expression. We investigated the role of JP in a model of SLE in MRL/lpr mice, and identified the possible mechanism of action. Mice were divided randomly into four groups: control, model, and two treatment groups. Sections of renal tissue were stained with hematoxylin and eosin (H&E). Histopathologic changes in the kidney were evaluated by light microscopy. T-helper (Th)17 cells were analyzed by flow cytometry. mRNA levels of interleukin (IL)-17, CaMK4, and RORγt were measured by reverse transcription polymerase chain reaction (RT-PCR). CaMK4 expression was assessed by Western blotting. The results showed that the percentages of Th17, IL-17, and RORγt in mice treated with JP were decreased remarkably compared to the model group (p < 0.05). Interestingly, a high CaMK4 expression was observed in the SLE mice, which was inhibited by JP. These results suggest that CaMK4 activity was increased in T cells from MRL/lpr mice compared with the control group. Our findings support the conclusion that the effects of JP on MRL/lpr mice may involve the regulation of CaMK4 overexpression in MRL/lpr mice.

  18. Atlas of transgenic Tet-Off Ca2+/calmodulin-dependent protein kinase II and prion protein promoter activity in the mouse brain.

    PubMed

    Odeh, Francis; Leergaard, Trygve B; Boy, Jana; Schmidt, Thorsten; Riess, Olaf; Bjaalie, Jan G

    2011-02-14

    Conditional transgenic mouse models are important tools for investigations of neurodegenerative diseases and evaluation of potential therapeutic interventions. A popular conditional transgenic system is the binary tetracycline-responsive gene (Tet-Off) system, in which the expression of the gene of interest depends on a tetracycline-regulatable transactivator (tTA) under the control of a specific promoter construct. The most frequently used Tet-Off promoter mouse lines are the Ca(2+)/calmodulin-dependent protein kinase II (CamKII) and prion protein (PrP) promoter lines, respectively. To target the regulated gene of interest to relevant brain regions, a priori knowledge about the spatial distribution of the regulated gene expression in the brain is important. Such distribution patterns can be investigated using double transgenic mice in which the promoter construct regulates a LacZ reporter gene encoding the marker β-galactosidase which can be histologically detected using its substrate X-gal. We have previously published an atlas showing the brain-wide expression mediated by the Tet-Off PrP promoter mouse line, but the distribution of activity in the Tet-Off CamKII promoter mouse line is less well known. To compare promoter activity distributions in these two Tet-Off mouse lines, we have developed an online digital atlas tailored for side-by-side comparison of histological section images. The atlas provides a comprehensive list of brain regions containing X-gal labeling and an interactive dual image viewer tool for panning and zooming of corresponding section images. Comparison of spatial expression patterns between the two lines show considerable regional and cellular differences, relevant in context of generation and analysis of inducible models based on these two tetracycline responsive promoter mouse lines. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. The inhibitor of calcium/calmodulin-dependent protein kinase II KN93 attenuates bone cancer pain via inhibition of KIF17/NR2B trafficking in mice.

    PubMed

    Liu, Yue; Liang, Ying; Hou, Bailing; Liu, Ming; Yang, Xuli; Liu, Chenglong; Zhang, Juan; Zhang, Wei; Ma, Zhengliang; Gu, Xiaoping

    2014-09-01

    The N-methyl-d-aspartate receptor (NMDAR) containing subunit 2B (NR2B) is critical for the regulation of nociception in bone cancer pain, although the precise molecular mechanisms remain unclear. KIF17, a kinesin motor, plays a key role in the dendritic transport of NR2B. The up-regulation of NR2B and KIF17 transcription results from an increase in phosphorylated cAMP-response element-binding protein (CREB), which is activated by calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we hypothesized that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. The expression of spinal t-CaMKII, p-CaMKII, NR2B and KIF17 after inoculation was also evaluated. These results showed that inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant up-regulation of p-CaMKII, NR2B and KIF17 expression after inoculation. Intrathecal administration of KN93, a CaMKII inhibitor, down-regulated these three proteins and attenuated bone cancer pain in a dose- and time-dependent manner. These findings indicated that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain, and inhibition of CaMKII may be a useful alternative or adjunct therapy for relieving cancer pain.

  20. Dysfunction of calcium/calmodulin/CaM kinase IIα cascades in the medial prefrontal cortex in post-traumatic stress disorder.

    PubMed

    Wen, Yu; Li, Bin; Han, Fang; Wang, Enhua; Shi, Yuxiu

    2012-11-01

    Post-traumatic stress disorder (PTSD) is a significant problem that may affect individuals who have been exposed to a traumatic event or events, including combat, violent crime or childhood abuse. The medial prefrontal cortex (mPFC) is known to be significantly involved in emotional adjustment, particularly introspection, amygdala inhibition and emotional memory. In the acute phase of severe traumatic stress, the mPFC appears to undergo a change in plasticity for a short time, which suggests that the mPFC may be the reponse-sensitizing region. Calcium (Ca2+) is one of most significant intracellular messengers; the appropriate concentration of Ca2+ is necessary for neuronal excitability. When the Ca2+ concentration increases, Ca2+, calmodulin (CaM) and CaM kinase IIα (CaMKIIα) combine together to form the Ca2+‑CaM‑CaMKIIα signaling pathway, which is important in the plasticity of the central nervous system, learning and memory, mind, behavior and other types of cognitive activities. Our team studied the changes in the Ca2+-CaM-CaMKIIα levels in the mPFC of rats following a single-prolonged stress (SPS). The SPS, a credible method for establishing a rat model of PTSD, has been internationally recognized. The free intracellular Ca2+ concentration in the mPFC in the PTSD group was significantly higher than that in the control group 1 day after SPS exposure (P<0.05) and decreased 7 days after SPS; CaM expression significantly increased, while CaMKIIα expression significantly decreased in the mPFC 1 day after SPS compared with the control group. These findings suggest dysfunction of the Ca2+-CaM-CaMKIIα cascades in the mPFC, which may relate to the pathogenesis of the abnormal functioning of the mPFC in PTSD.

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

    PubMed

    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.

  2. Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association

    SciTech Connect

    Ozaki, Hana; Katoh, Tsuyoshi; Nakagawa, Ryoko; Ishihara, Yasuhiro; Sueyoshi, Noriyuki; Kameshita, Isamu; Taniguchi, Takanobu; Hirano, Tetsuo; Yamazaki, Takeshi; Ishida, Atsuhiko

    2016-09-02

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation. - Highlights: • NFL was identified as a CaMKP-binding protein in an insoluble fraction of rat brain. • CaMKP bound to NFL in solution with a K{sub d} value of 73 ± 17 nM. • A CaMKP-NFL complex was found in NGF-differentiated PC12 cells. • CaMKP-binding to NFL inhibited its filament association. • CaMKP may regulate network formation of neurofilaments in neurons.

  3. Calcium-calmodulin-dependent kinase II modulates Kv4.2 channel expression and upregulates neuronal A-type potassium currents.

    PubMed

    Varga, Andrew W; Yuan, Li-Lian; Anderson, Anne E; Schrader, Laura A; Wu, Gang-Yi; Gatchel, Jennifer R; Johnston, Daniel; Sweatt, J David

    2004-04-07

    Calcium-calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K(+) channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K(+) current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K(+) channels.

  4. Age-dependent changes in autophosphorylation of alpha calcium/calmodulin dependent kinase II in hippocampus and amygdala after contextual fear conditioning.

    PubMed

    Fang, Ton; Kasbi, Kamillia; Rothe, Stephanie; Aziz, Wajeeha; Giese, K Peter

    2017-09-01

    The hippocampus and amygdala are essential brain regions responsible for contextual fear conditioning (CFC). The autophosphorylation of alpha calcium-calmodulin kinase II (αCaMKII) at threonine-286 (T286) is a critical step implicated in long-term potentiation (LTP), learning and memory. However, the changes in αCaMKII levels with aging and training in associated brain regions are not fully understood. Here, we studied how aging and training affect the levels of phosphorylated (T286) and proportion of phosphorylated:total αCaMKII in the hippocampus and amygdala. Young and aged mice, naïve (untrained) and trained in CFC, were analysed by immunohistochemistry for the levels of total and phosphorylated αCaMKII in the hippocampus and amygdala. We found that two hours after CFC training, young mice exhibited a higher level of phosphorylated and increased ratio of phosphorylated:total αCaMKII in hippocampal CA3 stratum radiatum. Furthermore, aged untrained mice showed a higher ratio of phosphorylated:total αCaMKII in the CA3 region of the hippocampus when compared to the young untrained group. No effect of training or aging were seen in the central, lateral and basolateral amygdala regions, for both phosphorylated and ratio of phosphorylated:total αCaMKII. These results show that aging impairs the training-induced upregulation of autophosphorylated (T286) αCaMKII in the CA3 stratum radiatum of the hippocampus. This indicates that distinct age-related mechanisms underlie CFC that may rely more heavily on NMDA receptor-dependent plasticity in young age. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Involvement of calcium-calmodulin protein kinase but not mitogen-activated protein kinase in light-induced phase delays and Per gene expression in the suprachiasmatic nucleus of the hamster.

    PubMed

    Yokota, S; Yamamoto, M; Moriya, T; Akiyama, M; Fukunaga, K; Miyamoto, E; Shibata, S

    2001-04-01

    It is known that Ca(2+)-dependent phosphorylation of cAMP response element binding protein (CREB) and the rapid induction of mPer1 and mPer2, mouse period genes in the suprachiasmatic nucleus (SCN) are associated with light-induced phase shifting. The CREB/CRE transcriptional pathway has been shown to be activated by calcium/calmodulin dependent kinase II (CaMKII) and mitogen-activated protein kinase (MAPK); however, there is a lack of evidence concerning whether the activation of CaMKII and/or MAPK elicited by photic stimuli are associated with the change in Per gene expression and behavioral phase shifting. In this experiment, we found there was an inhibitory effect by KN93, CaMKII inhibitor, on hamster Per1 and Per2 expression in the SCN and on phase delays in wheel running rhythm induced by light pulses. PD98059 and U0126, MAPK kinase inhibitors, however, affected neither light-induced Per1 and Per2 expression nor behavioral phase delays, even though PD98059 attenuated the light-induced phosphorylation of MAPK in the SCN. The present findings demonstrate that the light-induced activation of CaMKII plays an important role in the induction of Per1 and Per2 mRNA in the hamster SCN as well as phase shifting. These results suggest that gated induction of Per1 and/or Per2 genes through CaMKII-CREB/CRE accompanied with photic stimuli may be a critical step in phase shifting.

  6. Activation of Calcium/Calmodulin-Dependent Protein Kinase II in Obesity Mediates Suppression of Hepatic Insulin Signaling

    PubMed Central

    Ozcan, Lale; de Souza, Jane Cristina; Harari, Alp Avi; Backs, Johannes; Olson, Eric N.; Tabas, Ira

    2013-01-01

    SUMMARY A hallmark of obesity is selective suppression of hepatic insulin signaling (“insulin resistance”), but critical gaps remain in our understanding of the molecular mechanisms. We now report a major role for hepatic CaMKII, a calcium-responsive kinase that is activated in obesity. Genetic targeting of hepatic CaMKII, its downstream mediator p38, or the p38 substrate and stabilizer MK2 enhances insulin-induced p-Akt in palmitate-treated hepatocytes and obese mouse liver, leading to metabolic improvement. The mechanism of improvement begins with induction of ATF6 and the ATF6 target p58IPK, a chaperone that suppresses the PERK—p-eIF2α— ATF4 branch of the UPR. The result is a decrease in the ATF target TRB3, an inhibitor of insulin-induced p-Akt, leading to enhanced activation of Akt and its downstream metabolic mediators. These findings increase our understanding of the molecular mechanisms linking obesity to selective insulin resistance and suggest new therapeutic targets for type 2 diabetes and metabolic syndrome. PMID:24268736

  7. Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter

    PubMed Central

    Steinkellner, Thomas; Yang, Jae-Won; Montgomery, Therese R.; Chen, Wei-Qiang; Winkler, Marie-Therese; Sucic, Sonja; Lubec, Gert; Freissmuth, Michael; Elgersma, Ype; Sitte, Harald H.; Kudlacek, Oliver

    2012-01-01

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP+) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKIIT305D), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP+ efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome. PMID:22778257

  8. Calcium/calmodulin-dependent protein kinase (CaMK) Iα mediates the macrophage inflammatory response to sepsis

    PubMed Central

    Zhang, Xianghong; Guo, Lanping; Collage, Richard D.; Stripay, Jennifer L.; Tsung, Allan; Lee, Janet S.; Rosengart, Matthew R.

    2011-01-01

    Dysregulated Ca2+ handling is prevalent during sepsis and postulated to perpetuate the aberrant inflammation underlying subsequent organ dysfunction and death. The signal transduction cascades mediating these processes are unknown. Here, we identify that CaMKIα mediates the Mφ response to LPS in vitro and the inflammation and organ dysfunction of sepsis in vivo. We show that LPS induced active pThr177-CaMKIα in RAW 264.7 cells and murine peritoneal Mφ, which if inhibited biochemically with STO609 (CaMKK inhibitor) or by RNAi, reduces LPS-induced production of IL-10. Transfection of constitutively active CaMKIα (CaMKI293), but not a kinase-deficient mutant (CaMKI293K49A), induces IL-10 release. This production of IL-10 is mediated by CaMKIα-dependent regulation of p38 MAPK activation. CaMKIα activity also mediates the cellular release of HMGB1 by colocalizing with and regulating the packaging of HMGB1 into secretory lysosomes. During endotoxemia, mice receiving in vivo CaMKIαRNAi display reduced systemic concentrations of IL-10 and HMGB1 in comparison with mice receiving NTRNAi. These data support the biological relevance of CaMKIα-dependent IL-10 production and HMGB1 secretion. In a CLP model of sepsis, CaMKIαRNAi mice display reduced systemic concentrations of IL-10, IL-6, TNF-α, and HMGB1 in comparison with NTRNAi mice, which correlate with reductions in the development of renal dysfunction. These data support that CaMKIα signaling is integral to the Mφ responding to LPS and may also be operant in vivo in regulating the inflammation and organ dysfunction consequent to sepsis. PMID:21372190

  9. Calcium/calmodulin-mediated signal network in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2003-01-01

    Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. Calmodulin, the predominant calcium receptor, is one of the best-characterized calcium sensors in eukaryotes. In recent years, completion of the Arabidopsis genome project and advances in functional genomics have helped to identify and characterize numerous calmodulin-binding proteins in plants. There are some similarities in Ca(2+)/calmodulin-mediated signaling in plants and animals. However, plants possess multiple calmodulin genes and many calmodulin target proteins, including unique protein kinases and transcription factors. Some of these proteins are likely to act as "hubs" during calcium signal transduction. Hence, a better understanding of the function of these calmodulin target proteins should help in deciphering the Ca(2+)/calmodulin-mediated signal network and its role in plant growth, development and response to environmental stimuli.

  10. Calcium/calmodulin-mediated signal network in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2003-01-01

    Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. Calmodulin, the predominant calcium receptor, is one of the best-characterized calcium sensors in eukaryotes. In recent years, completion of the Arabidopsis genome project and advances in functional genomics have helped to identify and characterize numerous calmodulin-binding proteins in plants. There are some similarities in Ca(2+)/calmodulin-mediated signaling in plants and animals. However, plants possess multiple calmodulin genes and many calmodulin target proteins, including unique protein kinases and transcription factors. Some of these proteins are likely to act as "hubs" during calcium signal transduction. Hence, a better understanding of the function of these calmodulin target proteins should help in deciphering the Ca(2+)/calmodulin-mediated signal network and its role in plant growth, development and response to environmental stimuli.

  11. Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

    PubMed

    Yilmaz, Mehtap; Gangopadhyay, Samudra S; Leavis, Paul; Grabarek, Zenon; Morgan, Kathleen G

    2013-02-07

    CaMKII (Ca²⁺/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²⁶, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, we generated a phosphospecific Ser²⁶ antibody and demonstrated an increase in Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, we mutated Ser²⁶ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²⁸⁷ autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²⁶ of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most probably by blocking ATP binding. We propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity.

  12. The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.

    PubMed

    Dhavan, Rani; Greer, Paul L; Morabito, Maria A; Orlando, Lianna R; Tsai, Li-Huei

    2002-09-15

    Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

  13. The fine-structural distribution of G-protein receptor kinase 3, beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl(-)-cotransporter in rodent olfactory epithelia.

    PubMed

    Menco, Bert Ph M

    2005-03-01

    The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na+, K(+)-2Cl(-)-cotransporter NKCC1 regulates Cl(-)-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to beta-arrestin-2, five to CaMKII (one to both the alpha and beta form, and two each specific to CaMKII alpha and beta), two to PDE1C2, and three to Cl(-)-cotransporters. Only antibodies to Cl(-)-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, beta- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.

  14. Structural basis for activation of calcineurin by calmodulin

    PubMed Central

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

    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 activates calmodulin-dependent protein kinases. Previously published data has hinted that the regulatory domain of calcineurin is intrinsically disordered. In this work we demonstrate that the regulatory domain is unstructured and that it folds upon binding calmodulin, ousting the autoinhibitory domain from the catalytic site. The regulatory domain is 95 residues long, with the autoinhibitory domain attached to its C-terminal end and the 24 residue calmodulin binding region towards the N-terminal end. This is unlike the calmodulin-dependent protein kinases which have calmodulin binding sites and autoinhibitory domains immediately adjacent in sequence. Our data demonstrate that not only does the calmodulin binding region fold, but that an ~25-30 residue region between it and the autoinhibitory domain also folds, resulting in over half of the regulatory domain adopting α-helical structure. This appears to be the first observation of calmodulin inducing folding of this scale outside of its binding site on a target protein. PMID:22100452

  15. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  16. Ca(2+)/Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy.

    PubMed

    Duran, Javier; Lagos, Daniel; Pavez, Mario; Troncoso, Mayarling F; Ramos, Sebastián; Barrientos, Genaro; Ibarra, Cristian; Lavandero, Sergio; Estrada, Manuel

    2017-01-01

    Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR)-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and myocyte-enhancer factor 2 (MEF2) play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX) rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg⋅kg(-1)⋅week(-1)) for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN) phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286) and phospholambam (PLN) (Thr17) in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that testosterone

  17. Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

    PubMed Central

    Findlay, W. A.; Gradwell, M. J.; Bayley, P. M.

    1995-01-01

    The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM. PMID:8563635

  18. Multivalent interactions of calcium/calmodulin-dependent protein kinase II with the postsynaptic density proteins NR2B, densin-180, and alpha-actinin-2.

    PubMed

    Robison, A J; Bass, Martha A; Jiao, Yuxia; MacMillan, Leigh B; Carmody, Leigh C; Bartlett, Ryan K; Colbran, Roger J

    2005-10-21

    Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.

  19. Membrane actions of 1α,25(OH)2D3 are mediated by Ca(2+)/calmodulin-dependent protein kinase II in bone and cartilage cells.

    PubMed

    Doroudi, Maryam; Plaisance, Marc C; Boyan, Barbara D; Schwartz, Zvi

    2015-01-01

    1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Calmodulin-dependent kinase 1beta is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro.

    PubMed

    Pedersen, Mona E; Fortunati, Dario; Nielsen, Marit; Brorson, Sverre-Henning; Lekva, Tove; Nissen-Meyer, Lise Sofie H; Gautvik, Vigdis T; Shahdadfar, Aboulghassem; Gautvik, Kaare M; Jemtland, Rune

    2008-10-01

    The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.

  1. Amino Acid Sequence of a Novel Calmodulin from the Unicellular Alga Chlamydomonas1

    PubMed Central

    Lukas, Thomas J.; Wiggins, Michael E.; Watterson, D. Martin

    1985-01-01

    An amino acid sequence for a Chlamydomonas calmodulin has been elucidated with emphasis on the characterization of differences that are unique to Chlamydomonas and Dictyostelium calmodulin. While the concentration of calmodulin required for half-maximal activation of plant NAD kinase varies among vertebrate, higher plant, algal, and slime mold calmodulins, only calmodulins from the unicellular alga Chlamydomonas and the slime mold Dictyostelium show increased maximal activation of NAD kinase (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798; Marshak, Clarke, Roberts, Watterson 1984 Biochemistry 23: 2891-2899). The same preparations of calmodulin do not show major differences in phosphodiesterase or myosin light chain kinase activator activity. We report here that a Chlamydomonas calmodulin has four primary structural features similar to Dictyostelium that are not found in other calmodulins characterized to date: an altered carboxy terminus including a novel 11-residue extension for Chlamydomonas calmodulin, unique residues at positions 81 and 118, and an unmethylated lysine at position 115. The only amino acid sequence identity unique to Chlamydomonas and Dictyostelium calmodulin is the presence of a lysine at position 115 instead of a trimethyllysine. These studies indicate that the methylation state of lysine 115 may be important in the maximal NAD kinase activator activity of calmodulin and support the concept that calmodulin has multiple functional domains in addition to multiple structural domains. PMID:16664269

  2. 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*

    PubMed Central

    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 both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel “double anchor” motif. Moreover, the orientation of the helical peptide on the surface of Ca2+-SCaM4 is distinct from other known complexes. The N-lobe of Ca2+-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca2+ are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex. PMID:19667066

  3. Calcium/calmodulin kinase inhibitors and immunosuppressant macrolides rapamycin and FK506 inhibit progestin- and glucocorticosteroid receptor-mediated transcription in human breast cancer T47D cells.

    PubMed

    Le Bihan, S; Marsaud, V; Mercier-Bodard, C; Baulieu, E E; Mader, S; White, J H; Renoir, J M

    1998-07-01

    The effects of immunosuppressants and inhibitors of specific calcium/calmodulin kinase (CaMK) of types II and IV on progestin/glucocorticosteroid-induced transcription were studied in two human stably transfected breast cancer T47D cell lines. The lines contain the chloramphenicol acetyl transferase (CAT) gene under control either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter containing five glucocorticosteroid/progestin hormone response elements [T47D-(GRE)5-CAT]. Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression was inhibited in a dose-dependent manner in both lines by preincubation with rapamycin (Rap) and, to a lesser extent, with FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcription. The antagonist RU486 inhibited all the effects of both progestin and TA, suggesting that progesterone receptor (PR)-, as well as glucocorticosteroid receptor (GR)- mediated transactivation are targets of immunosuppressants and CaMKs in T47D cells. Indeed, Northern analysis showed that Rap, KN62, and, to a lesser degree, FK506 inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Addition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, these data indicate that Rap and FK506, as well as CaMK inhibitors, inhibit steroid-induced activities of exogenous, as well as of some endogenous, steroid receptor-regulated genes by a mechanism preceding hormone-induced receptor activation. Rap appeared to stabilize a 9S form of [3H]Org 2058-PR complexes isolated from T47D (GRE)5CAT cell nuclei. By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity

  4. Detection of calmodulin-binding proteins using a 32P-labeled GST-calmodulin fusion protein and a novel renaturation protocol.

    PubMed

    Fischer, R; Wei, Y; Berchtold, M

    1996-08-01

    To identify calmodulin-binding proteins in cellular extracts and tissue homogenates and to analyze purified calmodulin target proteins, overlay procedures using 125I-calmodulin or, more recently, nonradioactive biotinylated calmodulin have been widely used. Here we describe a rapid, alternative method for detecting calmodulin-binding proteins with a 32P-labeled calmodulin probe generated as a glutathione-S-transferase (GST)-fusion protein. We used a modified pGEX-2TK vector, which contains the flag epitope and the consensus sequence R-R-A-S, that can be phosphorylated by the cAMP-dependent protein kinase A. The fusion protein is easily purified from bacterial bysates by affinity chromatography using glutathione-Sepharose 4B beads. Phosphorylation of GST-calmodulin is performed directly on the beads and, after elution with reduced glutathione, the labeled calmodulin probe can be used for overlay experiments. We also describe a rapid renaturation protocol that enhances the signal for some but not all calmodulin-binding proteins and is used after the proteins have been transferred to nitrocellulose filters. Furthermore, we have compared the specificity and sensitivity of the 32P-labeled GST-calmodulin overlay with those of 125I-calmodulin and biotinylated calmodulin, clearly indicating that our newly developed protocol is a suitable alternative to conventionally used calmodulin overlay procedures.

  5. Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.

    PubMed Central

    Roberts, D M; Besl, L; Oh, S H; Masterson, R V; Schell, J; Stacey, G

    1992-01-01

    Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants. Images PMID:1325656

  6. Administration of a Ca-super(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor prevents the learning deficit observed in spinal rats after noncontingent shock administration.

    PubMed

    Baumbauer, Kyle M; Young, Erin E; Hoy, Kevin C; Abood, Athena; Joynes, Robin L

    2007-06-01

    Research has shown that spinal rats given shock to the hind leg when it is in an extended position (contingent shock) will learn to maintain a flexion response. However, subjects that experience shock irrespective of leg position (noncontingent shock) do not exhibit this learning. The current studies examined the role of Ca-super(2+)/calmodulin-dependent protein kinase II (CaMKII) in this learning deficit. Subjects were given intrathecal injections of CaMKII inhibitor solution or artificial cerebrospinal fluid (aCSF) 15 min prior to and immediately or 4 hr following noncontingent shock training. Results demonstrate that the CaMKII inhibitor successfully reversed the learning deficit when injected prior to and immediately following training. These results indicate the importance of CaMKII in the learning deficit present in spinal animals trained with noncontingent shock. Copyright (c) 2007 APA, all rights reserved.

  7. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  8. The pepper calmodulin gene CaCaM1 is involved in reactive oxygen species and nitric oxide generation required for cell death and the defense response.

    PubMed

    Choi, Hyong Woo; Lee, Dong Hyuk; Hwang, Byung Kook

    2009-11-01

    Calcium signaling has emerged as an important signal transduction pathway of higher plants in response to biotic and abiotic stresses. Ca2+-bound calmodulin (CaM) plays a critical role in decoding and transducing stress signals by activating specific targets. Here, we isolated and functionally characterized the pathogen-responsive CaM gene, Capsicum annuum calmodulin 1 (CaCaM1), from pepper (C. annuum) plants. The cellular function of CaCaM1 was verified by Agrobacterium spp.-mediated transient expression in pepper and transgenic overexpression in Arabidopsis thaliana. Agrobacterium spp.-mediated transient expression of CaCaM1 activated reactive oxygen species (ROS), nitric oxide (NO) generation, and hypersensitive response (HR)-like cell death in pepper leaves, ultimately leading to local acquired resistance to Xanthomonas campestris pv. vesicatoria. CaCaM1-overexpression (OX) Arabidopsis exhibited enhanced resistance to Pseudomonas syringae and Hyaloperonospora parasitica, which was accompanied by enhanced ROS and NO generation and HR-like cell death. Treatment with the calcium-channel blocker suppressed the oxidative and NO bursts and HR-like cell death that were triggered by CaCaM1 expression in pepper and Arabidopsis, suggesting that calcium influx is required for the activation of CaCaM1-mediated defense responses in plants. Upon treatment with the CaM antagonist, virulent P. syringae pv. tomato-induced NO generation was also compromised in CaCaM1-OX leaves. Together, these results suggest that the CaCaM1 gene functions in ROS and NO generation are essential for cell death and defense responses in plants.

  9. A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress.

    PubMed

    Sun, Xiaoli; Yang, Shanshan; Sun, Mingzhe; Wang, Sunting; Ding, Xiaodong; Zhu, Dan; Ji, Wei; Cai, Hua; Zhao, Chaoyue; Wang, Xuedong; Zhu, Yanming

    2014-05-01

    It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein-protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress.

  10. Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide

    SciTech Connect

    Roth, S.M.; Schneider, D.M.; Strobel, L.A.; Wand, A.J. Univ. of Illinois, Urbana ); Van Berkum, M.F.A.; Means, A.R. )

    1992-02-11

    The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive {sup 1}H and {sup 15}N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with {alpha}-{sup 15}N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly {sup 15}N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C{sub {alpha}}H, and C{sub {beta}}H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.

  11. Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin.

    PubMed

    Osborn, Kenneth D; Bartlett, Ryan K; Mandal, Abhijit; Zaidi, Asma; Urbauer, Ramona J Bieber; Urbauer, Jeffrey L; Galeva, Nadya; Williams, Todd D; Johnson, Carey K

    2004-10-12

    We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.

  12. Structure and dynamics of calmodulin in solution.

    PubMed Central

    Wriggers, W; Mehler, E; Pitici, F; Weinstein, H; Schulten, K

    1998-01-01

    To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides. PMID:9545028

  13. Protein kinase D activity controls endothelial nitric oxide synthesis.

    PubMed

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. © 2014. Published by The Company of Biologists Ltd.

  14. Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain*♦

    PubMed Central

    Yokom, Adam L.; Morishima, Yoshihiro; Lau, Miranda; Su, Min; Glukhova, Alisa; Osawa, Yoichi; Southworth, Daniel R.

    2014-01-01

    Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca2+-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis. PMID:24737326

  15. Calcium/calmodulin dependent protein kinase II regulates the phosphorylation of cyclic AMP-responsive element-binding protein of spinal cord in rats following noxious stimulation.

    PubMed

    Fang, Li; Wu, Jing; Zhang, Xuan; Lin, Qing; Willis, William D

    2005-02-01

    We have previously reported that intradermal capsaicin injection causes the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats. The present study was designed to investigate the role of calcium/camodulin protein dependent protein kinase II (CaM kinase II) in the regulation of phosphorylation of CREB after capsaicin injection. We found that capsaicin injection produces a significant upregulation of phosphorylated CREB in the spinal cord of rat. Intrathecal treatment with a CaM kinase II inhibitor, KN-93, significantly blocked the increased phosphorylation of CREB, but did not affect the CREB protein itself. These results suggest that increased phosphorylation of CREB protein may contribute to central sensitization following acute peripheral noxious stimuli, and the effect may be regulated through the activation of CaM kinase cascades.

  16. Distinct Roles for Mitogen-Activated Protein Kinase Signaling and CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 in Regulating the Peak Time and Amplitude of the Plant General Stress Response1[W][OPEN

    PubMed Central

    Bjornson, Marta; Benn, Geoffrey; Song, Xingshun; Comai, Luca; Franz, Annaliese K.; Dandekar, Abhaya M.; Drakakaki, Georgia; Dehesh, Katayoon

    2014-01-01

    To survive environmental challenges, plants have evolved tightly regulated response networks, including a rapid and transient general stress response (GSR), followed by well-studied stress-specific responses. The mechanisms underpinning the GSR have remained elusive, but a functional cis-element, the rapid stress response element (RSRE), is known to confer transcription of GSR genes rapidly (5 min) and transiently (peaking 90–120 min after stress) in vivo. To investigate signal transduction events in the GSR, we used a 4xRSRE:LUCIFERASE reporter in Arabidopsis (Arabidopsis thaliana), employing complementary approaches of forward and chemical genetic screens, and identified components regulating peak time versus amplitude of RSRE activity. Specifically, we identified a mutant in CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) with reduced RSRE activation, verifying this transcription factor’s role in activation of the RSRE-mediated GSR. Furthermore, we isolated a mutant in MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) KINASE KINASE1 (mekk1-5), which displays increased basal and an approximately 60-min earlier peak of wound-induced RSRE activation. The double mekk1/camta3 mutant positioned CAMTA3 downstream of MEKK1 and verified their distinct roles in GSR regulation. mekk1-5 displays programmed cell death and overaccumulates reactive oxygen species and salicylic acid, hallmarks of the hypersensitive response, suggesting that the hypersensitive response may play a role in the RSRE phenotype in this mutant. In addition, chemical inhibition studies suggest that the MAPK network is required for the rapid peak of the RSRE response, distinguishing the impact of chronic (mekk1-5) from transient (chemical inhibition) loss of MAPK signaling. Collectively, these results reveal underlying regulatory components of the plant GSR and further define their distinct roles in the regulation of this key biological process. PMID:25157030

  17. Abnormal changes in voltage-gated sodium channels Na(V)1.1, Na(V)1.2, Na(V)1.3, Na(V)1.6 and in calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats.

    PubMed

    Xu, Xiaoxue; Guo, Feng; Lv, Xintong; Feng, Rui; Min, Dongyu; Ma, Lihua; Liu, Yajing; Zhao, Jinsheng; Wang, Lei; Chen, Tianbao; Shaw, Chris; Hao, Liying; Cai, Jiqun

    2013-07-01

    Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

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

    PubMed

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Peroxisomal plant nitric oxide synthase (NOS) protein is imported by peroxisomal targeting signal type 2 (PTS2) in a process that depends on the cytosolic receptor PEX7 and calmodulin.

    PubMed

    Corpas, Francisco J; Barroso, Juan B

    2014-06-05

    Nitric oxide (NO) production in plant peroxisomes by l-arginine-dependent NO synthase activity has been proven. The PEX5 and PEX7 PTS receptors, which recognize PTS1- and PTS2-containing proteins, are localized in the cytosol. Using AtPex5p and AtPex7p knockdown in Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i, we found that the l-arginine-dependent protein responsible for NO generation in peroxisomes appears to be imported through an N-terminal PTS2. Pharmacological analyzes using a calcium channel blocker and calmodulin (CaM) antagonist show that the import of the peroxisomal NOS protein also depends on calcium and calmodulin.

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

    PubMed

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

    2016-01-01

    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.

  1. Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach.

    PubMed

    Al-Shboul, Othman; Mustafa, Ayman

    2015-06-01

    Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzyme-linked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress.

  2. Calcium/Calmodulin-Mediated Gravitropic Response in Plants

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.

    2002-01-01

    The goal of this project was to gain a fundamental understanding of how calcium/calmodulin-mediated signaling is involved in gravity signal transduction in plants. During the period of support, significant progress was made in elucidating the role of calmodulin and its target proteins in gravitropism. This laboratory has made breakthroughs by cloning and characterizing genes that are involved in calcium/calmodulin-mediated signaling. Some of these genes show altered expression under hypergravity and simulated microgravity conditions. A major advance was made in our attempts to understand gravity signal transduction by cloning and characterizing a catalase which requires calcium/calmodulin for its activation. Our results suggest that calcium/calmodulin have dual roles in regulating the level of hydrogen peroxide (H202), a signal molecule that plays a major role in gravitropism. It is well established that auxin plays a major role in gravitropism. Our results indicate that there is a 'cross-talk' between calcium/calmodulin-mediated signaling and auxin-mediated signal transduction. Auxin-regulated SAUR proteins that are involved in gravitropism bind to calmodulin in a calcium-dependent manner. A novel chimeric calcium/calmodulin-dependent protein kinase was cloned and characterized and its role in gravity signal transduction was investigated. These studies have provided some answers to the fundamental questions about how signal molecules such as calcium, H202, and hormones such as auxin bring about the ultimate gravitropic response and the integral role of calmodulin in gravity signal transduction. This NASA-funded study has led to some spinoffs that have applications in solving agricultural problems. The Washington State University Research Foundation has obtained several patents related to this work.

  3. The Phosphatase-Resistant Isoform of CaMKI, Ca²⁺/Calmodulin-Dependent Protein Kinase Iδ (CaMKIδ), Remains in Its "Primed" Form without Ca²⁺ Stimulation.

    PubMed

    Senga, Yukako; Ishida, Atsuhiko; Shigeri, Yasushi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2015-06-16

    Ca²⁺/calmodulin-dependent protein kinase I (CaMKI) is known to play pivotal roles in Ca²⁺ signaling pathways. Four isoforms of CaMKI (α, β, γ, and δ) have been reported so far. CaMKI is activated through phosphorylation by the upstream kinase, CaMK kinase (CaMKK), and phosphorylates downstream targets. When CaMKI was transiently expressed in 293T cells, CaMKIα was not phosphorylated at all under low-Ca²⁺ conditions in the cells. In contrast, we found that CaMKIδ was significantly phosphorylated and activated to phosphorylate cAMP response element-binding protein (CREB) under the same conditions. Herein, we report that the sustained activation of CaMKIδ is ascribed to its phosphatase resistance resulting from the structure of its N-terminal region. First, we examined whether CaMKIδ is more readily phosphorylated by CaMKK than CaMKIα, but no significant difference was observed. Next, to compare the phosphatase resistance between CaMKIα and CaMKIδ, we assessed the dephosphorylation of the phosphorylated CaMKIs by CaMK phosphatase (CaMKP/PPM1F). Surprisingly, CaMKIδ was hardly dephosphorylated by CaMKP, whereas CaMKIα was significantly dephosphorylated under the same conditions. To date, there have been no detailed reports concerning dephosphorylation of CaMKI. Through extensive analysis of CaMKP-catalyzed dephosphorylation of various chimeric and point mutants of CaMKIδ and CaMKIα, we identified the amino acid residues responsible for the phosphatase resistance of CaMKIδ (Pro-57, Lys-62, Ser-66, Ile-68, and Arg-76). These results also indicate that the phosphatase resistance of CaMKI is largely affected by only several amino acids in its N-terminal region. The phosphatase-resistant CaMKI isoform may play a physiological role under low-Ca²⁺ conditions in the cells.

  4. Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor.

    PubMed

    Asano, Takayuki; Kunieda, Noriko; Omura, Yuhi; Ibe, Hirokazu; Kawasaki, Tsutomu; Takano, Makoto; Sato, Miho; Furuhashi, Hideyuki; Mujin, Toshiyuki; Takaiwa, Fumio; Wu Cy, Chuan-yin; Tada, Yuichi; Satozawa, Tomomi; Sakamoto, Masahiro; Shimada, Hiroaki

    2002-03-01

    Suc, an end product of photosynthesis, is metabolized by Suc synthase in sink organs as an initial step in the biosynthesis of storage products. Suc synthase activity is known to be regulated by reversible phosphorylation, but the details of this process are unclear at present. Rice SPK, a calcium-dependent protein kinase, is expressed uniquely in the endosperm of immature seed, and its involvement in the biosynthetic pathways of storage products was suggested. Antisense SPK transformants lacked the ability to accumulate storage products such as starch, but produced watery seed with a large amount of Suc instead, as the result of an inhibition of Suc degradation. Analysis of in vitro phosphorylation indicated that SPK phosphorylated specifically a Ser residue in Suc synthase that has been shown to be important for its activity in the degradation of Suc. This finding suggests that SPK is involved in the activation of Suc synthase. It appears that SPK is a Suc synthase kinase that may be important for supplying substrates for the biosynthesis of storage products.

  5. Ca2+–calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

    PubMed Central

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-01-01

    Abstract Recent studies have demonstrated that changes in the activity of calcium–calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation–contraction (E–C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM–GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+–CaMKII–DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII. PMID:21486818

  6. Moderate Alcohol Drinking and the Amygdala Proteome: Identification and Validation of Calcium/Calmodulin Dependent Kinase II and AMPA Receptor Activity as Novel Molecular Mechanisms of the Positive Reinforcing Effects of Alcohol

    PubMed Central

    Salling, Michael C.; Faccidomo, Sara P.; Li, Chia; Psilos, Kelly; Galunas, Christina; Spanos, Marina; Agoglia, Abigail E.; Kash, Thomas L.; Hodge, Clyde W.

    2015-01-01

    BACKGROUND Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood. METHODS Here, we conducted a high-through put screen of the amygdala proteome in mice after moderate alcohol drinking (n = 12/group) followed by behavioral studies (n = 6–8/group) to uncover novel molecular mechanisms of the positive reinforcing properties of alcohol that strongly influence the development of addiction. RESULTS Two-dimensional difference in-gel electrophoresis with matrix assisted laser desorption ionization tandem time-of-flight identified 29 differentially expressed proteins in the amygdala of nondependent C57BL/6J mice following 24 days of alcohol drinking. Alcohol-sensitive proteins included calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and a network of functionally linked proteins that regulate neural plasticity and glutamate-mediated synaptic activity. Accordingly, alcohol drinking increased α-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) in central amygdala (CeA) and phosphorylation of AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in CeA and lateral amygdala. Further, CaMKIIα-Thr286 and GluA1-Ser831 phosphorylation was increased in CeA and lateral amygdala of mice that lever-pressed for alcohol versus the nondrug reinforcer sucrose. Mechanistic studies showed that targeted pharmacologic inhibition of amygdala CaMKII or AMPAR activity specifically inhibited the positive reinforcing properties of alcohol but not sucrose. CONCLUSIONS Moderate alcohol drinking increases the activity and function of plasticity-linked protein networks in the amygdala that regulate the positive reinforcing effects of the drug. Given the prominence of positive reinforcement in the etiology of addiction, we propose that alcohol-induced adaptations in CaMKIIα and AMPAR signaling in the amygdala may serve as a molecular gateway from use to abuse. PMID:25579851

  7. Calcium/calmodulin-dependent serine protein kinase (CASK), a protein implicated in mental retardation and autism-spectrum disorders, interacts with T-Brain-1 (TBR1) to control extinction of associative memory in male mice

    PubMed Central

    Huang, Tzyy-Nan; Hsueh, Yi-Ping

    2017-01-01

    Background Human genetic studies have indicated that mutations in calcium/calmodulin-dependent serine protein kinase (CASK) result in X-linked mental retardation and autism-spectrum disorders. We aimed to establish a mouse model to study how Cask regulates mental ability. Methods Because Cask encodes a multidomain scaffold protein, a possible strategy to dissect how CASK regulates mental ability and cognition is to disrupt specific protein–protein interactions of CASK in vivo and then investigate the impact of individual specific protein interactions. Previous in vitro analyses indicated that a rat CASK T724A mutation reduces the interaction between CASK and T-brain-1 (TBR1) in transfected COS cells. Because TBR1 is critical for glutamate receptor, ionotropic, N-methyl-d-aspartate receptor subunit 2B (Grin2b) expression and is a causative gene for autism and intellectual disability, we then generated CASK T740A (corresponding to rat CASK T724A) mutant mice using a gene-targeting approach. Immunoblotting, coimmunoprecipitation, histological methods and behavioural assays (including home cage, open field, auditory and contextual fear conditioning and conditioned taste aversion) were applied to investigate expression of CASK and its related proteins, the protein–protein interactions of CASK, and anatomic and behavioural features of CASK T740A mice. Results The CASK T740A mutation attenuated the interaction between CASK and TBR1 in the brain. However, CASK T740A mice were generally healthy, without obvious defects in brain morphology. The most dramatic defect among the mutant mice was in extinction of associative memory, though acquisition was normal. Limitations The functions of other CASK protein interactions cannot be addressed using CASK T740A mice. Conclusion Disruption of the CASK and TBR1 interaction impairs extinction, suggesting the involvement of CASK in cognitive flexibility. PMID:28234597

  8. Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice.

    PubMed

    Jackson, Kia J; Imad Damaj, M

    2013-02-15

    Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the β2-selective antagonist dihydro-β-erythroidine (DHβE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20min prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHβE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through β2-containing nicotinic receptors.

  9. Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

    PubMed

    Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

    2016-07-15

    In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  10. Low Dose Ultraviolet B Irradiation Increases Hyaluronan Synthesis in Epidermal Keratinocytes via Sequential Induction of Hyaluronan Synthases Has1–3 Mediated by p38 and Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Signaling*

    PubMed Central

    Rauhala, Leena; Hämäläinen, Lasse; Salonen, Pauliina; Bart, Geneviève; Tammi, Markku; Pasonen-Seppänen, Sanna; Tammi, Raija

    2013-01-01

    Hyaluronan, a major epidermal extracellular matrix component, responds strongly to different kinds of injuries. This also occurs by UV radiation, but the mechanisms involved are poorly understood. The effects of a single ultraviolet B (UVB) exposure on hyaluronan content and molecular mass, and expression of genes involved in hyaluronan metabolism were defined in monolayer and differentiated, organotypic three-dimensional cultures of rat epidermal keratinocytes. The signals regulating the response were characterized using specific inhibitors and Western blotting. In monolayer cultures, UVB increased hyaluronan synthase Has1 mRNA already 4 h postexposure, with a return to control level by 24 h. In contrast, Has2 and Has3 were persistently elevated from 8 h onward. Silencing of Has2 and especially Has3 decreased the UVB-induced accumulation of hyaluronan. p38 and Ca2+/calmodulin-dependent protein kinase II pathways were found to be involved in the UVB-induced up-regulation of Has2 and Has3 expression, respectively, and their inhibition reduced hyaluronan deposition. However, the expressions of the hyaluronan-degrading enzymes Hyal1 and Hyal2 and the hyaluronan receptor Cd44 were also up-regulated by UVB. In organotypic cultures, UVB treatment also resulted in increased expression of both Has and Hyal genes and shifted hyaluronan toward a smaller size range. Histochemical stainings indicated localized losses of hyaluronan in the epidermis. The data show that exposure of keratinocytes to acute, low dose UVB increases hyaluronan synthesis via up-regulation of Has2 and Has3. The simultaneously enhanced catabolism of hyaluronan demonstrates the complexity of the UVB-induced changes. Nevertheless, enhanced hyaluronan metabolism is an important part of the adaptation of keratinocytes to radiation injury. PMID:23645665

  11. Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel alpha(1C)-subunit gene (Cacna1c) by DREAM translocation.

    PubMed

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-06-01

    Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

  12. Analysis of the state of posttranslational calmodulin methylation in developing pea plants. [Pisum sativum

    SciTech Connect

    Oh, Sukheung; Roberts, D.M. )

    1990-07-01

    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 ({sup 3}H)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.

  13. IL-1β induces GFAP expression in vitro and in vivo and protects neurons from traumatic injury-associated apoptosis in rat brain striatum via NFκB/Ca²⁺-calmodulin/ERK mitogen-activated protein kinase signaling pathway.

    PubMed

    Sticozzi, C; Belmonte, G; Meini, A; Carbotti, P; Grasso, G; Palmi, M

    2013-11-12

    Reactive astrogliosis, a feature of neuro-inflammation is induced by a number of endogenous mediators including cytokines. Despite interleukin-1 beta (IL-1β) stands out as the major inducer of this process, the underlying mechanism and its role on neuronal viability remain elusive. We investigated in human astrocytoma cells and the rat brain striatum, the role of the nuclear factor-kB (NF-kB) intracellular Ca(2+) concentration ([Ca(2+)]i) calmodulin (CaM) and extracellular regulated mitogen-activated protein kinases (ERK1/2) in IL-1β-induced expression of glial fibrillary acidic protein (GFAP) and neuronal apoptosis associated to a brain trauma. Cell data showed that IL-1β (1 ng/ml) increased NF-kB, pERK1/2 and GFAP expression. Nevertheless, further increase in IL-1β levels reversed progressively these responses. Preventing ERK1/2 activation with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]-butadiene antagonized IL-1β-induced GFAP expression while inhibiting selectively nuclear translocation of NF-kB with caffeic-acid phenethyl-ester down-regulated both ERK1/2 and GFAP expression induced by IL-1β. The GFAP response was also prevented by antagonizing selectively increase in [Ca(2+)]i, CaM activity or inducible nitric oxide synthase expression with respectively ryanodine plus 2-aminoethoxydiphenyl-borate, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride and N-[(3-(aminomethyl)-phenyl]methyl]-ethanimidamide dihydrochloride. Data in vivo supported these findings and showed that GFAP expression induced by IL-1β (50 ng/ml) correlated with attenuated glial scar formation and reduced neuronal apoptosis. Our data identified the NF-kB/Ca(2+)-CaM/ERK signaling pathway as a novel in vivo key regulator of IL-1β-induced astrogliosis which may represent a potential target in neurodegeneration.

  14. Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize [Phosphorylation of a NAC Transcription Factor by ZmCCaMK Regulates Abscisic Acid-Induced Antioxidant Defense in Maize

    SciTech Connect

    Zhu, Yuan; Yan, Jingwei; Liu, Weijuan; Liu, Lei; Sheng, Yu; Sun, Yue; Li, Yanyun; Scheller, Henrik Vibe; Jiang, Mingyi; Hou, Xilin; Ni, Lan; Zhang, Aying

    2016-05-10

    Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize, which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 display a partially overlapping expression pattern with ZmCCaMK after ABA treatment and H2O2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates S113 of ZmNAC84 in vitro, and S113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco can improve drought tolerance, and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H2O2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK, and subsequently the activated ZmCCaMK phosphorylates ZmNAC84 at S113, thereby inducing antioxidant defense by activating downstream genes.

  15. Phosphorylation of a NAC Transcription Factor by a Calcium/Calmodulin-Dependent Protein Kinase Regulates Abscisic Acid-Induced Antioxidant Defense in Maize [Phosphorylation of a NAC Transcription Factor by ZmCCaMK Regulates Abscisic Acid-Induced Antioxidant Defense in Maize

    DOE PAGES

    Zhu, Yuan; Yan, Jingwei; Liu, Weijuan; ...

    2016-05-10

    Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize, which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 display a partially overlapping expression pattern with ZmCCaMK after ABA treatment and H2O2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates S113 of ZmNAC84 in vitro, andmore » S113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco can improve drought tolerance, and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H2O2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK, and subsequently the activated ZmCCaMK phosphorylates ZmNAC84 at S113, thereby inducing antioxidant defense by activating downstream genes.« less

  16. Ca(2+)/calmodulin-dependent protein kinase IIα (αCaMKII) controls the activity of the dopamine transporter: implications for Angelman syndrome.

    PubMed

    Steinkellner, Thomas; Yang, Jae-Won; Montgomery, Therese R; Chen, Wei-Qiang; Winkler, Marie-Therese; Sucic, Sonja; Lubec, Gert; Freissmuth, Michael; Elgersma, Ype; Sitte, Harald H; Kudlacek, Oliver

    2012-08-24

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.

  17. Rapid 1α,25(OH)₂D ₃ membrane-mediated activation of Ca²⁺/calmodulin-dependent protein kinase II in growth plate chondrocytes requires Pdia3, PLAA and caveolae.

    PubMed

    Doroudi, Maryam; Boyan, Barbara D; Schwartz, Zvi

    2014-08-01

    1α,25-Dihydroxy vitamin D3 [1α,25(OH)2D3] regulates growth zone chondrocytes (GC) via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. 1α,25(OH)2D3 initiates its membrane effects via its specific membrane-associated receptor (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating PLA2, resulting in prostaglandin E2 (PGE2) release and protein kinase C activation. Recently, we reported that 1α,25(OH)2D3 rapidly activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in GC cells. However, the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3-dependent rapid activation of CaMKII are not clear. The aim of the present study was to evaluate the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3 membrane-stimulated CaMKII activation. Pre-treating chondrocytes from the growth zone of the rat costochondral cartilage with antibodies against PLAA or Pdia3 blocked activation of CaMKII by 1α,25(OH)2D3. PLAA peptide rapidly activated CaMKII in GC cells. Caveolae disruption abolished CaMKII activation in response to 1α,25(OH)2D3 or PLAA peptide treatment. Immunoprecipitation studies showed increased CaM binding to PLAA in response to 1α,25(OH)2D3. The results indicated that Pdia3, PLAA and caveolae are required for rapid 1α,25(OH)2D3 membrane-mediated activation of CaMKII. 1α,25(OH)2D3 signaling via Pdia3 receptor triggered the interaction between PLAA and CaM suggesting that CaM may play a major role linking PLAA to CaMKII in membrane-mediated actions of 1α,25(OH)2D3.

  18. Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases.

    PubMed

    Walkup, Ward G; Washburn, Lorraine; Sweredoski, Michael J; Carlisle, Holly J; Graham, Robert L; Hess, Sonja; Kennedy, Mary B

    2015-02-20

    synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases*

    PubMed Central

    Walkup, Ward G.; Washburn, Lorraine; Sweredoski, Michael J.; Carlisle, Holly J.; Graham, Robert L.; Hess, Sonja; Kennedy, Mary B.

    2015-01-01

    synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons. PMID:25533468

  20. Laminar shear stress upregulates endothelial Ca²⁺-activated K⁺ channels KCa2.3 and KCa3.1 via a Ca²⁺/calmodulin-dependent protein kinase kinase/Akt/p300 cascade.

    PubMed

    Takai, Jun; Santu, Alexandra; Zheng, Haifeng; Koh, Sang Don; Ohta, Masanori; Filimban, Linda M; Lemaître, Vincent; Teraoka, Ryutaro; Jo, Hanjoong; Miura, Hiroto

    2013-08-15

    In endothelial cells (ECs), Ca²⁺-activated K⁺ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca²⁺ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca²⁺/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca²⁺/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm²) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8-30), whereas oscillatory shear (OS; ± 5 dyn/cm² × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

  1. Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.

    PubMed Central

    Romero, I; Maldonado, A M; Eraso, P

    1997-01-01

    Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of approximately 1 microM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein. PMID:9148755

  2. Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways.

    PubMed

    Bodnárová, Michaela; Martásek, Pavel; Moroz, Leonid L

    2005-04-01

    We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a

  3. Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

    PubMed

    Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

    2012-11-01

    Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation.

  4. Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells.

    PubMed

    Heise, Nicole; Palme, Daniela; Misovic, Milan; Koka, Saisudha; Rudner, Justine; Lang, Florian; Salih, Helmut R; Huber, Stephan M; Henke, Guido

    2010-01-01

    Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

  5. Food restriction increases NMDA receptor-mediated calcium-calmodulin kinase II and NMDA receptor/extracellular signal-regulated kinase 1/2-mediated cyclic amp response element-binding protein phosphorylation in nucleus accumbens upon D-1 dopamine receptor stimulation in rats.

    PubMed

    Haberny, S L; Carr, K D

    2005-01-01

    Biological drive states exert homeostatic control in part by increasing the reinforcing effects of environmental incentive stimuli. An apparent by-product of this adaptive response is the enhanced acquisition of drug self-administration behavior in food-restricted (FR) animals. While previous research has demonstrated increased central sensitivity to rewarding effects of abused drugs and direct dopamine (DA) receptor agonists in FR subjects, the underlying neurobiology is not well understood. Recently, it was demonstrated that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958 produces a stronger activation of striatal extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element-binding protein (CREB) in FR relative to ad libitum (AL) fed rats. The main purpose of the present study was to characterize the involvement and mechanisms of interaction between NMDA receptor function and the augmented cellular responses to D-1 DA receptor stimulation in nucleus accumbens (NAc) of FR rats. In experiment 1, Western immunoblotting was used to demonstrate that i.c.v. injection of SKF-82958 (20 microg) produces greater phosphorylation of the NMDA NR1 subunit and calcium-calmodulin kinase II (CaMK II) in NAc of FR as compared with AL rats. In experiment 2, pretreatment of subjects with the NMDA antagonist, MK-801 (1.0 mg/kg, i.p.) decreased SKF-82958-induced activation of CaMK II, ERK1/2 and CREB, and reversed the augmenting effect of FR on activation of all three proteins. In experiment 3, pretreatment with the mitogen-activated protein kinase/ERK kinase inhibitor SL-327 (60 mg/kg, i.p.) suppressed SKF-82958- induced activation of ERK1/2 and reversed the augmenting effect of FR on CREB activation. These results point to specific neuroadaptations in the NAc of FR rats whereby D-1 DA receptor stimulation leads to increased NMDA NR1 subunit phosphorylation and consequent increases in NMDA receptor-dependent CaMK II and ERK1

  6. Correlation between calmodulin activity and gravitropic sensitivity in primary roots of maize

    NASA Technical Reports Server (NTRS)

    Stinemetz, C. L.; Kuzmanoff, K. M.; Evans, M. L.; Jarrett, H. W.

    1987-01-01

    Recent evidence indicates a role for calcium and calmodulin in the gravitropic response of primary roots of maize (Zea mays, L.). We examined this possibility by testing the relationship between calmodulin activity and gravitropic sensitivity in roots of the maize cultivars Merit and B73 x Missouri 17. Roots of the Merit cultivar require light to the gravitropically competent. The gravitropic response of the Missouri cultivar is independent of light. The occurrence of calmodulin in primary roots of these maize cultivars was tested by affinity gel chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with bovine brain calmodulin as standard. The distribution of calmodulin activity was measured using both the phosphodiesterase and NAD kinase assays for calmodulin. These assays were performed on whole tissue segments, crude extracts, and purified extracts. In light-grown seedlings of the Merit cultivar or in either dark- or light-grown seedlings of the Missouri cultivar, calmodulin activity per millimeter of root tissue was about 4-fold higher in the apical millimeter than in the subtending 3 millimeters. Calmodulin activity was very low in the apical millimeter of roots of dark-grown (gravitropically nonresponsive) seedlings of the Merit cultivar. Upon illumination, the calmodulin activity in the apical millimeter increased to a level comparable to that of light-grown seedlings and the roots became gravitropically competent. The time course of the development of gravitropic sensitivity following illumination paralleled the time course of the increase in calmodulin activity in the apical millimeter of the root. The results are consistent with the suggestion that calmodulin plays an important role in the gravitropic response of roots.

  7. Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.) 1

    PubMed Central

    Perera, Imara Y.; Zielinski, Raymond E.

    1992-01-01

    The expression of calmodulin mRNA and protein were measured during a growth cycle of carrot (Daucus carota L.) cells grown in suspension culture. A full-length carrot calmodulin cDNA clone isolated from a λgt10 library was used to measure steady-state calmodulin mRNA levels. During the exponential phase of culture growth when mitotic activity and oxidative respiration rates were maximal, calmodulin mRNA levels were 4- to 5-fold higher than they were during the later stages of culture growth, when respiration rates were lower and growth was primarily by cell expansion. Net calmodulin polypeptide synthesis, as measured by pulse-labeling in vivo with [35S]methionine, paralleled the changes in calmodulin steady-state mRNA level during culture growth. As a consequence, net calmodulin polypeptide synthesis declined 5- to 10-fold during the later stages of culture growth. The qualitative spectrum of polypeptides synthesized and accumulated by the carrot cells during the course of a culture cycle, however, remained largely unchanged. Calmodulin polypeptide levels, in contrast to its net synthesis, remained relatively constant during the exponential phases of the culture growth cycle and increased during the later stages of culture growth. Our data are consistent with increased calmodulin polypeptide turnover associated with periods of rapid cell proliferation and high levels of respiration. Images Figure 1 Figure 2 Figure 4 PMID:16653062

  8. Activation of ERK1/2 and TNF-α production are regulated by calcium/calmodulin signaling pathway during Penicillium marneffei infection within human macrophages.

    PubMed

    Chen, Renqiong; Ji, Guangquan; Wang, Ling; Ren, Hong; Xi, Liyan

    2016-04-01

    Previous study have shown that Penicillium marneffei (P. marneffei)-induced TNF-α production via an extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase-dependent mechanism is an important host defence mechanism against P. marneffei in human macrophages. Therefore, we explore signaling pathway that regulates TNF-α secretion and activation of ERK1/2 by intracellular signaling mechanisms during P. marneffei infection. We found that ERK1/2 activation was dependent on the calcium/calmodulin/calmodulin kinase Ⅱ pathway in P. marneffei-infected human macrophages. In contrast, P. marneffei-induced p38 MAPK activation was negatively regulated by calcium/calmodulin/calmodulin kinase Ⅱ signaling pathway. Furthermore, TNF-α production in P. marneffei-infected human macrophages was also dependent on Ca(2+)/calmodulin/calmodulin kinase Ⅱ pathway. These data suggest that Ca(2+)/calmodulin/calmodulin kinase Ⅱ pathway plays vital regulatory roles in macrophage activation and subsequent cytokine production during P. marneffei infection.

  9. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells.

    PubMed

    Cheng, A; Chan, S L; Milhavet, O; Wang, S; Mattson, M P

    2001-11-16

    Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome c release, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.

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

    PubMed Central

    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 nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei. PMID:7823935

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

    SciTech Connect

    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.

  12. Calcium and calcium-dependent protein kinases are involved in nitric oxide- and auxin-induced adventitious root formation in cucumber.

    PubMed

    Lanteri, María Luciana; Pagnussat, Gabriela Carolina; Lamattina, Lorenzo

    2006-01-01

    A few years ago it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. In the present study, the involvement of Ca2+ and the regulation of Ca2+-dependent protein kinase (CDPK) activity during IAA- and NO-induced AR formation was evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca2+ channel inhibitors and Ca2+ chelators in affecting AR formation induced by IAA or NO was also examined. Results indicate that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca2+ pools. Protein extracts from cucumber hypocotyls were assayed for CDPK activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This CDPK activity was affected in both extracts from NO- and IAA-treated explants in the presence of the specific NO-scavenger cPTIO, suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA-induced AR formation, were inhibited by calmodulin antagonists. Furthermore, the induction of CDPK activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase.

  13. Functional expression of chicken calmodulin in yeast.

    PubMed

    Ohya, Y; Anraku, Y

    1989-01-31

    The coding region of a chicken calmodulin cDNA was fused to a galactose-inducible GAL1 promoter, and an expression system was constructed in the yeast Saccharomyces cerevisiae. Expression of calmodulin was demonstrated by purifying the heterologously expressed protein and analyzing its biochemical properties. When the expression plasmid was introduced into a calmodulin gene (cmd1)-disrupted strain of yeast, the cells grew in galactose medium, showing that chicken calmodulin could complement the lesion of yeast calmodulin functionally. Repression of chicken calmodulin in the (cmd1)-disrupted strain caused cell cycle arrest with a G2/M nucleus, as observed previously with a conditional-lethal mutant of yeast calmodulin. These results suggest that the essential function of calmodulin for cell proliferation is conserved in cells ranging from yeast to vertebrate cells.

  14. [Ca2+ (calmodulin)-dependent phosphorylation of plasma membranes of pig myometrium].

    PubMed

    Prishchepa, L A; Kondratiuk, T P; Kurskiĭ, M D

    1989-01-01

    The high-purified vesicles of pig myometrium sarcolemma closed, mainly, so that the cytoplasmatic side is outside possess the Ca2+ (calmodulin)-dependent protein kinase activity. The initial rate of the endogenic phosphorylation without exogenic calmodulin is 6.3 and with its presence--10.7 pmol of 32Pi 1 min per 1 mg of protein. Km for ATP is equal to 164 microM, and Vmax--0.27 nmol of 32Pi 1 min per 1 mg of protein. Exogenic calmodulin increases the affinity to ATP (50 microM), Vmax being unchanged. Under optimal concentrations of calmodulin (10(-7)-10(-6) M) and 10(-4) M Ca2+ the protein kinase activity is 0.132 nmol of 32Pi min per 1 mg of protein. Electrophoresis in DS-PAAG has shown that membrane proteins with molecular weight of 105, 58, 25, 12 and 2 kDa are basic substrates of Ca2+ (calmodulin)-dependent phosphorylation. Trifluoperazine++ in the concentration of 40 microM inhibits phosphorylation of all five proteins. Ca2+ (calmodulin)-dependent phosphorylation is supposed to be a regulator of Ca2+-transport processes of sarcolemma.

  15. Role of Calmodulin in Cell Proliferation

    NASA Technical Reports Server (NTRS)

    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.

  16. Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia.

    PubMed

    Moore, Claire E J; Mikolajek, Halina; Regufe da Mota, Sergio; Wang, Xuemin; Kenney, Justin W; Werner, Jörn M; Proud, Christopher G

    2015-05-01

    Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

  17. Oxidative stress in cardiovascular diseases and obesity: role of p66Shc and protein kinase C.

    PubMed

    De Marchi, Elena; Baldassari, Federica; Bononi, Angela; Wieckowski, Mariusz R; Pinton, Paolo

    2013-01-01

    Reactive oxygen species (ROS) are a byproduct of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis. An imbalance between ROS production and the cellular antioxidant defence system leads to oxidative stress. Environmental factors and genetic interactions play key roles in oxidative stress mediated pathologies. In this paper, we focus on cardiovascular diseases and obesity, disorders strongly related to each other; in which oxidative stress plays a fundamental role. We provide evidence of the key role played by p66(Shc) protein and protein kinase C (PKC) in these pathologies by their intracellular regulation of redox balance and oxidative stress levels. Additionally, we discuss possible therapeutic strategies aimed at attenuating the oxidative damage in these diseases.

  18. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    SciTech Connect

    Watkins, D.T. )

    1991-08-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing (32P)ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules.

  19. Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase.

    PubMed

    Zmijewski, Jaroslaw W; Banerjee, Sami; Bae, Hongbeom; Friggeri, Arnaud; Lazarowski, Eduardo R; Abraham, Edward

    2010-10-22

    Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5'-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H(2)O(2) was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1-312), we found that mutation of cysteine 299 to alanine diminished the ability of H(2)O(2) to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H(2)O(2) on kinase activity. Similar to the results obtained with H(2)O(2)-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H(2)O(2) are increased. These results demonstrate that physiologically relevant concentrations of H(2)O(2) can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.

  20. Shewanella oneidensis MR-1 H-NOX Regulation of a Histidine Kinase by Nitric Oxide

    PubMed Central

    Price, Mark S.; Chao, Lily; Marletta, Michael A.

    2008-01-01

    Nitric oxide (NO) signaling in animals controls processes such as smooth muscle relaxation and neurotransmission by activation of soluble guanylate cyclase (sGC). Prokaryotic homologs of the sGC heme domain, called H-NOX domains, have been identified and are generally found in a predicted operon in conjunction with a histidine kinase. Here, we show that an H-NOX protein (SO2144) from Shewanella oneidensis, directly interacts with the sensor histidine kinase (SO2145), binds NO in a 5-coordinate complex similar to mammalian sGC, and in that form inhibits the activity of a histidine kinase (SO2145). We also describe the first account of NO formation by S. oneidensis under anaerobic growth conditions derived from nitrate and nitrite. These observations suggest that the S. oneidensis H-NOX and histidine kinase pair function as part of a novel two-component signaling pathway that is responsive to NO formation from higher nitrogen oxides used as electron acceptors when oxygen is low and thereby functioning as an environmental sensor. PMID:17988156

  1. Computational comparison of a calcium-dependent jellyfish protein (apoaequorin) and calmodulin-cholesterol in short-term memory maintenance.

    PubMed

    Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K

    2017-03-06

    Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca(2+)-binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca(2+) ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca(2+) ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca(2+)-binding sites. Only calmodulin pore-lining regions contain Ca(2+) binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca(2+) channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to

  2. The role of AMP-activated protein kinase in mitochondrial biogenesis.

    PubMed

    Reznick, Richard M; Shulman, Gerald I

    2006-07-01

    While it has been known for more than 75 years that physical activity is associated with increased mitochondrial content in muscle, the molecular mechanism for this adaptive process has only recently been elucidated. This brief review examines existing studies that have identified AMPK-activated protein kinase (AMPK) and several other key regulators of mitochondrial biogenesis, including peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta, calcium/calmodulin-dependent protein kinase IV, and nitric oxide. In addition, the potential role of mitochondrial dysfunction in the pathogenesis of insulin resistance associated with ageing and type 2 diabetes mellitus is also discussed.

  3. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    PubMed

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.

  4. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances

    PubMed Central

    Howell, Stanley C.; Richards, David H.; Mitch, William A.; Wilson, Corey J.

    2016-01-01

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections, and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary-structure, protein hydrodynamics and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance. Namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  5. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

    PubMed Central

    Paul, A; Pendreigh, R H; Plevin, R

    1995-01-01

    1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. Images Figure 2 PMID:7533621

  6. Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

    PubMed Central

    Themanns, Madeleine; Mueller, Kristina M.; Kessler, Sonja M.; Golob-Schwarzl, Nicole; Mohr, Thomas; Kaltenecker, Doris; Bourgeais, Jerome; Paier-Pourani, Jamile; Friedbichler, Katrin; Schneller, Doris; Schlederer, Michaela; Zebedin-Brandl, Eva; Terracciano, Luigi M.; Han, Xiaonan; Kenner, Lukas; Wagner, Kay-Uwe; Mikulits, Wolfgang; Kozlov, Andrey V.; Heim, Markus H.; Gouilleux, Fabrice; Haybaeck, Johannes; Moriggl, Richard

    2016-01-01

    Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage. PMID:27713471

  7. Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation.

    PubMed

    Job, D; Rauch, C T; Fischer, E H; Margolis, R L

    1983-07-01

    Cold-labile microtubule protein can be rendered cold-stable by addition of a fraction containing a small number of polypeptides that are derived from cold-stable microtubules. These polypeptides can be obtained from purified cold-stable microtubules by passage through a DEAE-cellulose (DE-52) ion exchange column from which they emerge in the first eluate fraction. The stabilizing activity of these proteins is abolished by phosphorylation catalyzed by two types of protein kinases, one dependent on calmodulin and the other independent of that regulatory protein. The calmodulin-dependent reaction appears to phosphorylate mainly two polypeptides, 56 and 72 kilodaltons; the reaction is blocked by trifluoperazine. The calmodulin-independent reaction appears to phosphorylate different cold-stable microtubule-associated proteins. That reaction is observed only in purified material obtained from vigorously homogenized brain tissue. Gently homogenization yields cold-stable microtubules that are responsive only to the calmodulin-dependent protein kinase. A distinguishing feature of the calmodulin-independent reaction is that it does not occur on polypeptides while they are bound to the microtubules.

  8. LRRK2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a Drosophila model: suppression by curcumin.

    PubMed

    Yang, Dejun; Li, Tianxia; Liu, Zhaohui; Arbez, Nicolas; Yan, Jianqun; Moran, Timothy H; Ross, Christopher A; Smith, Wanli W

    2012-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is believed to involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause genetic forms of PD, and the LRRK2 locus contributes to sporadic PD. Environmental toxins are believed to act in part by causing oxidative stress. Here we employed cell and Drosophila models to investigate the interaction between LRRK2 genetic mutations and oxidative stress. We found that H(2)O(2) increased LRRK2 kinase activity and enhanced LRRK2 cell toxicity in cultured cells and mouse primary cortical neurons. Furthermore, a sub-toxic dose of H(2)O(2) significantly shortened the survival of LRRK2 transgenic flies and augmented LRRK2-induced locomotor defects and dopamine neuron loss. Treatment with a LRRK2 kinase inhibitor (GW5074) or an anti-oxidant (curcumin) significantly suppressed these PD-like phenotypes in flies. Moreover, curcumin significantly reduced LRRK2 kinase activity and the levels of oxidized proteins, and thus acted as not only an antioxidant but also a LRRK2 kinase inhibitor. These results indicate that LRRK2 genetic alterations can interact with oxidative stress, converging on a pathogenic pathway that may be related to PD. These studies also identified curcumin as a LRRK2 kinase inhibitor that may be a useful candidate for LRRK2-linked PD intervention.

  9. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

    PubMed

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  10. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    SciTech Connect

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  11. Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

    PubMed Central

    He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J.; Lu, H. Peter

    2015-01-01

    Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS. PMID:26311846

  12. A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

    NASA Technical Reports Server (NTRS)

    Porter Moore, C.; Zhang, J. Z.; Hamilton, S. L.

    1999-01-01

    Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

  13. Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

    PubMed Central

    Zmijewski, Jaroslaw W.; Banerjee, Sami; Bae, Hongbeom; Friggeri, Arnaud; Lazarowski, Eduardo R.; Abraham, Edward

    2010-01-01

    Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status. PMID:20729205

  14. Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development.

    PubMed

    Al-Shanti, Nasser; Stewart, Claire E

    2009-11-01

    The loss of muscle mass with age and disuse has a significant impact on the physiological and social well-being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross-sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age-related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin-like growth factor I (IGF-I) and Ca(2+)/calmodulin-dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF-I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca(2+)/calmodulin-dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T-cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca(2+) sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca(2+)/calmodulin-dependent kinases

  15. Calmodulin regulates KCNQ2 function in epilepsy

    PubMed Central

    Zhou, Xuhong; Zhuang, Fei; Li, Hong; Zheng, Kun; Hong, Ze; Feng, Weijing; Zhou, Wendi; Chen, Jian

    2016-01-01

    Epilepsy is linked to mutations in KCNQ channels. KCNQ channels including KCNQ2 and KCNQ3 are enriched in neurons, regulating action potential generation and modulation. Here, we showed that properties of KCNQ2 channel in rat hippocampal cultured neurons are regulated by ubiquitous calcium sensor calmodulin. We analyzed calmodulin function on the KCNQ2 channel in both HEK293 cells and neurons. We used shRNAs to suppress expression of calmodulin protein. On the other hand, we used cDNA to over-express calmodulin in HEK293 and neuron cells. In wild type and mis-sense mutations of KCNQ2 proteins, calmodulin over-expression enhanced outward K+ current and decreased neuronal activity. Meanwhile, calmodulin knockdown reduced KCNQ2 current and increased neuronal activity, showing that hippocampal neuronal excitability is regulated by expression level of calmodulin protein. Our data suggest that calmodulin performs a major function in regulating KCNQ2 properties via direct binding to KCNQ2 protein, indicating that calmodulin could be a target of as gene therapy in epilepsy. PMID:28078031

  16. CaM Kinase II-dependent pathophysiological signalling in endothelial cells.

    PubMed

    Cai, Hua; Liu, Depei; Garcia, Joe G N

    2008-01-01

    Calcium/calmodulin-dependent protein kinase II (CaM Kinase II) is a known modulator of cardiac pathophysiology. The present review uniquely focuses on novel CaM Kinase II-mediated endothelial cell signalling which, under pathophysiological conditions, may indirectly modulate cardiac functions via alterations in endothelial or endocardial responses. CaM Kinase II has four different isoforms and various splicing variants for each isoform. The endothelial cell CaM Kinase II isoforms are sensitive to KN93 and a threonine 286-mutated inhibitory peptide. In macrovascular endothelial cells derived from aortas, CaM Kinase II mediates redox-sensitive upregulation of endothelial nitric oxide synthase (eNOS) gene expression by hydrogen peroxide (H2O2) and oscillatory shear stress, and a rapid activation of eNOS in response to bradykinin. In endothelial cells derived from lung microvessels, CaM Kinase II mediates barrier dysfunction, particularly when activated by thrombin. In brain capillary endothelial cells, CaM Kinase II lies upstream of voltage-gated potassium channels and hypoxia-induced cell swelling. In both macrovascular and microvascular endothelial cells, CaM Kinase II mediates actin cytoskeleton reorganization via distinct p38 MAPK/HSP27 and ERK1/2/MLCK signalling pathways, respectively. Although understanding of endothelium-specific CaM Kinase II signalling is nascent, data accumulated so far have demonstrated a potentially significant role of CaM Kinase II in endothelial cell pathophysiology.

  17. Microfluidic free-flow electrophoresis for the discovery and characterisation of calmodulin binding partners

    NASA Astrophysics Data System (ADS)

    Herling, Therese; Linse, Sara; Knowles, Tuomas

    2015-03-01

    Non-covalent and transient protein-ligand interactions are integral to cellular function and malfunction. Key steps in signalling and regulatory pathways rely on reversible non-covalent protein-protein binding or ion chelation. Here we present a microfluidic free-flow electrophoresis method for detecting and characterising protein-ligand interactions in solution. We apply this method to probe the binding equilibria of calmodulin, a central protein to calcium signalling pathways. In this study we characterise the specific binding of calmodulin to phosphorylase kinase, a known target, and creatine kinase, which we identify as a putative binding partner through a protein array screen and surface plasmon resonance experiments. We verify the interaction between calmodulin and creatine kinase in solution using free-flow electrophoresis and investigate the effect of calcium and sodium chloride on the calmodulin-ligand binding affinity in free solution without the presence of a potentially interfering surface. Our results demonstrate the general applicability of quantitative microfluidic electrophoresis to characterise binding equilibria between biomolecules in solution.

  18. Conformational heterogeneity of the calmodulin binding interface

    NASA Astrophysics Data System (ADS)

    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.

  19. Conformational heterogeneity of the calmodulin binding interface

    PubMed Central

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

    2016-01-01

    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. PMID:27040077

  20. Conformational Frustration in Calmodulin-Target Recognition

    PubMed Central

    Tripathi, Swarnendu; Wang, Qian; Zhang, Pengzhi; Hoffman, Laurel; Waxham, M. Neal; Cheung, Margaret S.

    2015-01-01

    Calmodulin (CaM) is a primary calcium (Ca2+) signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM-binding domains from two Ca2+-CaM dependent kinases, CaMKI and CaMKII, by employing experimentally-constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM-CaMKII complex than the CaM-CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C-domain of CaM that need to be broken before the formation of intermolecular contacts with the N-domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations. PMID:25622562

  1. Calmodulin Methionine Residues are Targets For One-Electron Oxidation by Hydroxyl Radicals: Formation of S therefore N three-electron bonded Radical Complexes

    SciTech Connect

    Nauser, Thomas; Jacoby, Michael E.; Koppenol, Willem H.; Squier, Thomas C.; Schoneich, Christian

    2005-02-01

    The one-electron (1e) oxidation of organic sulfides and methionine (Met) constitutes an important reaction mechanism in vivo.1,2 Evidence for a Cu(II)-catalyzed oxidation of Met35 in the Alzheimer's disease -amyloid peptide was obtained,3 and, based on theoretical studies, Met radical cations were proposed as intermediates.4 In the structure of -amyloid peptide, the formation of Met radical cations appears to be facilitated by a preexisting close sulfur-oxygen (S-O) interaction between the Met35 sulfur and the carbonyl oxygen of the peptide bond C-terminal to Ile31.5 Substitution of Ile31 with Pro31 abolishes this S-O interaction,5 significantly reducing the ability of -amyloid to reduce Cu(II), and converts the neurotoxic wild-type -amyloid into a non-toxic peptide.6 The preexisting S-O bond characterized for wild-type -amyloid suggests that electron transfer from Met35 to Cu(II) is supported through stabilization of the Met radical cation by the electron-rich carbonyl oxygen, generating an SO-bonded7 sulfide radical cation (Scheme 1, reaction 1).5

  2. Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents.

    PubMed Central

    Knebel, A; Rahmsdorf, H J; Ullrich, A; Herrlich, P

    1996-01-01

    Several non-physiologic agents such as radiation, oxidants and alkylating agents induce ligand-independent activation of numerous receptor tyrosine kinases (RTKs) and of protein tyrosine kinases at the inner side of the plasma membrane (e.g. Dévary et al., 1992; Sachsenmaier et al., 1994; Schieven et al., 1994; Coffer et al., 1995). Here we show additional evidence for the activation of epidermal growth factor receptor (EGFR), and we show activation of v-ErbB, ErbB2 and platelet-derived growth factor receptor. As a common principle of action the inducing agents such as UVC, UVB, UVA, hydrogen peroxide and iodoacetamide inhibit receptor tyrosine dephosphorylation in a thiol-sensitive and, with the exception of the SH-alkylating agent, reversible manner. EGFR dephosphorylation can also be modulated by these non-physiologic agents in isolated plasma membranes in the presence of Triton X-100. Further, substrate (EGFR) and phosphatase have been separated: a membrane preparation of cells that have been treated with epidermal growth factor (EGF) and whose dephosphorylating enzymes have been permanently destroyed by iodoacetamide can be mixed with a membrane preparation from untreated cells which re-establishes EGFR dephosphorylation. This dephosphorylation can be modulated in vitro by UV and thiol agents. We conclude that RTKs exhibit significant spontaneous protein kinase activity; several adverse agents target (an) essential SH-group(s) carried by (a) membrane-bound protein tyrosine phosphatase(s). Images PMID:8895576

  3. Correlating Calmodulin Landscapes with Chemical Catalysis in Neuronal Nitric Oxide Synthase using Time-Resolved FRET and a 5-Deazaflavin Thermodynamic Trap

    PubMed Central

    2016-01-01

    A major challenge in enzymology is the need to correlate the dynamic properties of enzymes with, and understand the impact on, their catalytic cycles. This is especially the case with large, multicenter enzymes such as the nitric oxide synthases (NOSs), where the importance of dynamics has been inferred from a variety of structural, single-molecule, and ensemble spectroscopic approaches but where motions have not been correlated experimentally with mechanistic steps in the reaction cycle. Here we take such an approach. Using time-resolved spectroscopy employing absorbance and Förster resonance energy transfer (FRET) and exploiting the properties of a flavin analogue (5-deazaflavin mononucleotide (5-dFMN)) and isotopically labeled nicotinamide coenzymes, we correlate the timing of CaM structural changes when bound to neuronal nitric oxide synthase (nNOS) with the nNOS catalytic cycle. We show that remodeling of CaM occurs early in the electron transfer sequence (FAD reduction), not at later points in the reaction cycle (e.g., FMN reduction). Conformational changes are tightly correlated with FAD reduction kinetics and reflect a transient “opening” and then “closure” of the bound CaM molecule. We infer that displacement of the C-terminal tail on binding NADPH and subsequent FAD reduction are the likely triggers of conformational change. By combining the use of cofactor/coenzyme analogues and time-resolved FRET/absorbance spectrophotometry, we show how the reaction cycles of complex enzymes can be simplified, enabling a detailed study of the relationship between protein dynamics and reaction cycle chemistry—an approach that can also be used with other complex multicenter enzymes. PMID:27563493

  4. LRRK2 enhances oxidative stress-induced neurotoxicity via its kinase activity.

    PubMed

    Heo, Hye Young; Park, Ji-Min; Kim, Cy-Hyun; Han, Baek Soo; Kim, Kwang-Soo; Seol, Wongi

    2010-02-15

    LRRK2 is an autosomal dominant gene whose mutations cause familial Parkinson's disease (PD). The LRRK2 protein contains a functional kinase and a GTPase domain. PD phenotypes caused by LRRK2 mutations are similar to those of idiopathic PD, implying that LRRK2 is an important participant in PD pathogenesis. Of LRRK2's PD-specific mutations, the G2019S is the most frequently observed one. Its over-expression is known to increase kinase activity and neurotoxicity compared to wild type (WT) LRRK2. Here, using a simple colorimetric cell viability assay, we analyzed LRRK2's neurotoxicity in dopaminergic SN4741 cells following treatment with hydrogen peroxide. When WT, G2019S, or empty vector was expressed in SN4741 cells, cell death was modestly and significantly increased in the order of G2019S>WT>vector. When these transfected cells were treated with hydrogen peroxide to mimic oxidative stress, cellular neurotoxicity was enhanced in the same order (i.e. G2019S>WT>vector). Moreover, incubation of SN4741 cells with conditioned medium from cells expressing G2019S and subjected to hydrogen peroxide treatment exhibited 10-15% more cell death than conditioned medium from cells transfected with vector or WT, suggesting that G2019S-expressing cells secrete a factor(s) affecting viability of neighboring cells. The kinase domain was mapped to be responsible for oxidative stress-induced neurotoxicity. In addition, over-expression of WT and G2019S LRRK2 lead to a weak, but significant, increase in intracellular reactive oxygen species (ROS) in the order of G2019S>WT as measured by DCFH-DA assay in both the presence and absence of H(2)O(2) treatment. Furthermore, in G2019S-expressing cells, co-expression of the anti-oxidant protein DJ-1 or ERK inhibitor treatment restored survival rate to a level similar to that of cells transfected with control vector under H(2)O(2) treatment. Taken together, our data suggest that the LRRK2 kinase domain increases the generation of ROS and causes

  5. Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633

    PubMed Central

    Chen, Ming; Yi, Bing; Zhu, Ni; Wei, Xin; Zhang, Guan-Xin; Huang, Shengdong; Sun, Jianxin

    2016-01-01

    Aims Posttranslational modification, such as phosphorylation, plays an essential role in regulating activation of endothelial NO synthase (eNOS). In the present study, we aim to determine whether eNOS could be phosphorylated and regulated by a novel serine/threonine–protein kinase Pim1 in vascular endothelial cells (ECs). Methods and results Using immunoprecipitation and protein kinase assays, we demonstrated that Pim1 specifically interacts with eNOS, which leads to a marked phosphorylation of eNOS at Ser-633 and increased production of nitric oxide (NO). Intriguingly, in response to VEGF stimulation, eNOS phosphorylation at Ser-633 exhibits two distinct phases: transient phosphorylation occurring between 0 and 60 min and sustained phosphorylation occurring between 2 and 24 h, which are mediated by the protein kinase A (PKA) and Pim1, respectively. Inhibiting Pim1 by either pharmacological inhibitor SMI-4a or the dominant-negative form of Pim1 markedly attenuates VEGF-induced tube formation, while Pim1 overexpression significantly increases EC tube formation and migration in an NO-dependent manner. Importantly, Pim1 expression and eNOS phosphorylation at Ser-633 were substantially decreased in high glucose-treated ECs and in the aorta of db/db diabetic mice. Increased Pim1 expression ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay. Conclusion Our findings demonstrate Pim1 as a novel kinase that is responsible for the phosphorylation of eNOS at Ser-633 and enhances EC sprouting of aortic rings from diabetic mice, suggesting that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies. PMID:26598507

  6. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    NASA Technical Reports Server (NTRS)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  7. Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL

    PubMed Central

    Choi, Soo-Ho; Wiesner, Philipp; Almazan, Felicidad; Kim, Jungsu; Miller, Yury I.

    2012-01-01

    Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk−/− macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program. PMID:22384232

  8. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    NASA Technical Reports Server (NTRS)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  9. Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

    2001-01-01

    Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

  10. Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

    2001-01-01

    Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

  11. Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line).

    PubMed

    Olmos, Enrique; Martínez-Solano, Juan R; Piqueras, Abel; Hellín, Eladio

    2003-01-01

    The rapid generation of H(2)O(2) by Cd(2+)-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H(2)O(2) has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H(2)O(2) induced by Cd(2+). These data suggest the involvement of an NADPH oxidase-like enzyme leading to H(2)O(2) production through O(2)(*-) dismutation by superoxide dismutase enzymes. To investigate the implication of Ca(2+) channels in a Cd(2+)-induced oxidative burst, different inhibitors of Ca(2+) channels were used. Only La(3+) totally inhibited the generation of H(2)O(2) induced by Cd(2+). However, verapamil and nifedipine, inhibitors of Ca(2+) channels, were not effective. Calmodulin or a Ca(2+)-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H(2)O(2) induced by Cd(2+), suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.

  12. The effects of weak extremely low frequency magnetic fields on calcium/calmodulin interactions.

    PubMed Central

    Hendee, S P; Faour, F A; Christensen, D A; Patrick, B; Durney, C H; Blumenthal, D K

    1996-01-01

    Mechanisms by which weak electromagnetic fields may affect biological systems are of current interest because of their potential health effects. Lednev has proposed an ion parametric resonance hypothesis (Lednev, 1991, Bioelectromagnetics, 12:71-75), which predicts that when the ac, frequency of a combined dc-ac magnetic field equals the cyclotron frequency of calcium, the affinity of calcium for calcium-binding proteins such as calmodulin will be markedly affected. The present study evaluated Lednev's theory using two independent systems, each sensitive to changes in the affinity of calcium for calmodulin. One of the systems used was the calcium/calmodulin-dependent activation of myosin light chain kinase, a system similar to that previously used by Lednev. The other system monitored optical changes in the binding of a fluorescent peptide to the calcium/calmodulin complex. Each system was exposed to a 20.9 microT static field superimposed on a 20.9 microT sinusoidal field over a narrow frequency range centered at 16 Hz, the cyclotron frequency of the unhydrated calcium ion. In contrast to Lednev's predictions, no significant effect of combined dc-ac magnetic fields on calcium/calmodulin interactions was indicated in either experimental system. PMID:8744329

  13. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

    PubMed

    Peuchant, Evelyne; Bats, Marie-Lise; Moranvillier, Isabelle; Lepoivre, Michel; Guitton, Jérôme; Wendum, Dominique; Lacombe, Marie-Lise; Moreau-Gaudry, François; Boissan, Mathieu; Dabernat, Sandrine

    2017-04-01

    NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

  14. Protein Kinase A Governs Oxidative Phosphorylation Kinetics and Oxidant Emitting Potential at Complex I

    PubMed Central

    Lark, Daniel S.; Reese, Lauren R.; Ryan, Terence E.; Torres, Maria J.; Smith, Cody D.; Lin, Chien-Te; Neufer, P. Darrell

    2015-01-01

    The mitochondrial electron transport system (ETS) is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC)/cyclic AMP (cAMP)/Protein kinase A (PKA) axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA) cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduced complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowered both the Km and Vmax for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS. PMID:26635618

  15. Protein Kinase A Governs Oxidative Phosphorylation Kinetics and Oxidant Emitting Potential at Complex I.

    PubMed

    Lark, Daniel S; Reese, Lauren R; Ryan, Terence E; Torres, Maria J; Smith, Cody D; Lin, Chien-Te; Neufer, P Darrell

    2015-01-01

    The mitochondrial electron transport system (ETS) is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC)/cyclic AMP (cAMP)/Protein kinase A (PKA) axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA) cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduced complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowered both the K m and V max for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS.

  16. Tau regulates the subcellular localization of calmodulin

    SciTech Connect

    Barreda, Elena Gomez de

    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.

  17. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase

    PubMed Central

    Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F.; Kanthasamy, Anumantha G.; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2014-01-01

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (–/–) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (–/–) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (–/–) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. PMID:22512859

  18. Myocardial protection evoked by hyperoxic exposure involves signaling through nitric oxide and mitogen activated protein kinases.

    PubMed

    Ruusalepp, Arno; Czibik, Gabor; Flatebø, Torun; Vaage, Jarle; Valen, Guro

    2007-07-01

    Hyperoxic exposure in vivo (> 95% oxygen) attenuates ischemia-reperfusion injury, but the signaling mechanisms of this cardioprotection are not fully determined. We studied a possible role of nitric oxide (NO) and mitogen activated protein kinases (MAPK) in hyperoxic protection. Mice (n = 7-9 in each group) were kept in normoxic or hyperoxic environments for 15 min prior to harvesting the heart and Langendorff perfusion with global ischemia (45 min) and reperfusion (60 min). Endpoints were cardiac function and infarct size. Additional hearts were collected to evaluate MAPK phosphorylation (immunoblot). The nitric oxide synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059 and the p38 MAPK inhibitor FR167653 were injected intraperitoneally before hyperoxia or normoxia. Hyperoxia improved postischemic functional recovery and reduced infarct size (p < 0.05). Hyperoxic exposure caused cardiac phosphorylation of the MAPK family members p38 and ERK1/2, but not JNK. L-NAME, PD98059 and FR167653 all reduced the protection afforded by hyperoxic exposure, but did not influence performance or infarction in hearts of normoxic mice. The hyperoxia-induced phosphorylation of ERK1/2 and p38 was reduced by L-NAME and both MAPK inhibitors. Nitric oxide triggers hyperoxic protection, and ERK1/2 and p38 MAPK are involved in signaling of protection against ischemia-reperfusion injury.

  19. Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ.

    PubMed

    Shin, Eun-Joo; Duong, Chu Xuan; Nguyen, Xuan-Khanh Thi; Li, Zhengyi; Bing, Guoying; Bach, Jae-Hyung; Park, Dae Hun; Nakayama, Keiichi; Ali, Syed F; Kanthasamy, Anumantha G; Cadet, Jean Lud; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2012-06-15

    This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    PubMed Central

    Han, Ming-lei; Liu, Guo-hua; Guo, Jin; Yu, Shu-juan; Huang, Jing

    2016-01-01

    Retinal ganglion cell (RGC) degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB)-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H2O2)-induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H2O2. Western blot assay showed that in H2O2 -damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H2O2 -induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H2O2 -induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway. PMID:27127489

  1. Iron-dependent oxidative inactivation with affinity cleavage of pyruvate kinase.

    PubMed

    Murakami, Keiko; Tsubouchi, Ryoko; Fukayama, Minoru; Qiao, Shanlou; Yoshino, Masataka

    2009-07-01

    Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg2+, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg2+ implies the identical binding sites of Fe2+ and Mg2+, but the cleavage sites were discriminated from the cofactor Mg2+-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe2+ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg2+ concentrations are responsible for the control of cellular respiration and glycolysis.

  2. DISPLACEMENT OF α-ACTININ FROM THE NMDA RECEPTOR NR1 C0 DOMAIN BY Ca2+/CALMODULIN PROMOTES CAMKII BINDING

    PubMed Central

    Merrill, Michelle A.; Malik, Zulfiqar; Akyol, Zeynep; Bartos, Jason A.; Leonard, A. Soren; Hudmon, Andy; Shea, Madeline A.; Hell, Johannes W.

    2008-01-01

    Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and α-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing α-actinin from NR1 C0 upon Ca2+ influx. We show that Ca2+-depleted calmodulin and α-actinin simultaneously bind to NR1 C0. Upon addition of Ca2+, calmodulin dislodges α-actinin. Either the N- or C-terminal half of calmodulin is sufficient for Ca2+-induced displacement of α-actinin. While α-actinin directly antagonizes CaMKII binding to NR1 C0, the addition of Ca2+/calmodulin shifts binding of NR1 C0 toward CaMKII by displacing α-actinin. Displacement of α-actinin results in the simultaneous binding of calmodulin and CaMKII to NR1 C0. Our results reveal an intricate mechanism whereby Ca2+ functions to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII. PMID:17602661

  3. Nitric oxide signalling in plants: interplays with Ca2+ and protein kinases.

    PubMed

    Courtois, Cécile; Besson, Angélique; Dahan, Jennifer; Bourque, Stéphane; Dobrowolska, Grazyna; Pugin, Alain; Wendehenne, David

    2008-01-01

    Much attention has been paid to nitric oxide (NO) research since its discovery as a physiological mediator of plant defence responses. In recent years, newer roles have been attributed to NO, ranging from root development to stomatal closure. The molecular mechanisms underlying NO action in plants are just begun to emerge. The currently available data illustrate that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.

  4. Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

    PubMed Central

    Higaki, Yasuki; Mikami, Toshio; Fujii, Nobuharu; Hirshman, Michael F.; Koyama, Katsuhiro; Seino, Tetsuya; Tanaka, Keitaro; Goodyear, Laurie J.

    2010-01-01

    We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 μmol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-L-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 μmol/l had no effect on ATP concentrations and did not increase the activities of either the α1 or α2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. PMID:18303121

  5. Linalool prevents oxidative stress activated protein kinases in single UVB-exposed human skin cells.

    PubMed

    Gunaseelan, Srithar; Balupillai, Agilan; Govindasamy, Kanimozhi; Ramasamy, Karthikeyan; Muthusamy, Ganesan; Shanmugam, Mohana; Thangaiyan, Radhiga; Robert, Beaulah Mary; Prasad Nagarajan, Rajendra; Ponniresan, Veeramani Kandan; Rathinaraj, Pierson

    2017-01-01

    Ultraviolet-B radiation (285-320 nm) elicits a number of cellular signaling elements. We investigated the preventive effect of linalool, a natural monoterpene, against UVB-induced oxidative imbalance, activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling in HDFa cells. We observed that linalool treatment (30 μM) prevented acute UVB-irradiation (20 mJ/cm2) mediated loss of activities of antioxidant enzymes in HDFa cells. The comet assay results illustrate that linalool significantly prevents UVB-mediated 8-deoxy guanosine formation (oxidative DNA damage) rather than UVB-induced cyclobutane pyrimidine (CPD) formation. This might be due to its ability to prevent UVB-induced ROS formation and to restore the oxidative imbalance of cells. This has been reflected in UVB-induced overexpression of MAPK and NF-κB signaling. We observed that linalool inhibited UVB-induced phosphorylation of ERK1, JNK and p38 proteins of MAPK family. Linalool inhibited UVB-induced activation of NF-κB/p65 by activating IκBa. We further observed that UVB-induced expression of TNF-α, IL6, IL-10, MMP-2 and MMP-9 was modulated by linalool treatment in HDFa cells. Thus, linalool protects the human skin cells from the oxidative damages of UVB radiation and modulates MAPK and NF-κB signaling in HDFa cells. The present findings substantiate that linalool may act as a photoprotective agent against UVB-induced skin damages.

  6. Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

    PubMed

    Cacas, Jean-Luc; Gerbeau-Pissot, Patricia; Fromentin, Jérôme; Cantrel, Catherine; Thomas, Dominique; Jeannette, Emmanuelle; Kalachova, Tetiana; Mongrand, Sébastien; Simon-Plas, Françoise; Ruelland, Eric

    2017-04-01

    Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [(33) P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

  7. The Effects of Hydrazines and Related Compounds on Calcium Calmodulin Regulated Synaptic Processes

    DTIC Science & Technology

    1983-07-01

    1REPORT NUMBER 2.GOVT ACCESSION NO. S. RECIPIENT’S CATALOG NUM0ER 4.TTE(and Subtitle) S. TYPE OF REPORT & PERIOD COVERED The Effects of Hydrazines and...4F -631 0 1984 E IS. KEY WORDS (Continue on reverse side if necessary and identify by block nunmber) Calmodulin, protein kinase, adenylate cyclase...hydrazines, organophosphates, C) calcium, synaptic processes, protein phosphorylation. 20. BSTRACT (Continue an reverse side it necessary mid Identify

  8. Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA

    PubMed Central

    Ray, William K.; Potters, Mark B.; Haile, January D.; Kennelly, Peter J.

    2015-01-01

    The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3ʹ,5ʹ-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A. PMID:28248264

  9. Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide.

    PubMed

    Jensen, Baard Olav; Selheim, Frode; Døskeland, Stein Ove; Gear, Adrian R L; Holmsen, Holm

    2004-11-01

    The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change.

  10. Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

    PubMed

    Numakawa, Yumiko; Numakawa, Tadahiro; Matsumoto, Tomoya; Yagasaki, Yuki; Kumamaru, Emi; Kunugi, Hiroshi; Taguchi, Takahisa; Niki, Etsuo

    2006-05-01

    The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.

  11. Sustained cutaneous vasoconstriction during and following cyrotherapy treatment: Role of oxidative stress and Rho kinase.

    PubMed

    Christmas, Kevin M; Patik, Jordan C; Khoshnevis, Sepideh; Diller, Kenneth R; Brothers, R Matthew

    2016-07-01

    Cryotherapy is a therapeutic technique using ice or cold water applied to the skin to reduce bleeding, inflammation, pain, and swelling following soft tissue trauma and injury. While beneficial, there are some side effects such as pronounced vasoconstriction and tissue ischemia that are sustained for hours post-treatment. This study tested the hypothesis that this vasoconstriction is mediated by 1) the Rho-kinase pathway and/or 2) elevated oxidative stress. 9 subjects were fitted with a commercially available cryotherapy unit with a water perfused bladder on the lateral portion of the right calf. Participants were instrumented with three microdialysis probes underneath the bladder. One site received lactated ringers (control site), one received the Rho-Kinase inhibitor Fasudil, and one received Ascorbic Acid. Skin temperature (Tskin) and cutaneous vascular conductance (CVC) was measured at each site. Subjects had 1°C water perfused through the bladder for 30min, followed by passive rewarming for 90min. Tskin fell from ~34°C to ~18.0°C during active cooling across all sites and this response was similar for all sites (P>0.05 for all comparisons). During passive rewarming Tskin rose to a similar degree in all sites (P>0.05 relative to the end of cooling). %CVC was reduced during active cooling in all sites; however, the magnitude of this response was blunted in the Fasudil site relative to control (P<0.001 for all comparisons) and min 25 and 30 of cooling in the Ascorbic Acid site (P<0.05). During passive rewarming %CVC at the control and Ascorbic Acid sites did not change such that values were similar to the end of cooling (P>0.05 for each comparison). %CVC at the Fasudil site remained elevated during passive rewarming such that values were higher compared to the control and Ascorbic Acid sites throughout the 90min of passive rewarming (P<0.001 main effect of Fasudil). These findings indicate that the Rho-kinase pathway contributes to pronounced vasoconstriction

  12. Sustained Cutaneous Vasoconstriction during and Following Cyrotherapy Treatment: Role of Oxidative Stress and Rho Kinase

    PubMed Central

    Christmas, Kevin M.; Patik, Jordan C.; Khoshnevis, Sepideh; Diller, Kenneth R.; Brothers, R. Matthew

    2016-01-01

    Cryotherapy is a therapeutic technique using ice or cold water applied to the skin to reduce bleeding, inflammation, pain, and swelling following soft tissue trauma and injury. While beneficial, there are some side effects such as pronounced vasoconstriction and tissue ischemia that are sustained for hours post-treatment. This study tested the hypothesis that this vasoconstriction is mediated by 1) the Rho-kinase pathway and/or 2) elevated oxidative stress. 9 subjects were fitted with a commercially available cryotherapy unit with a water perfused bladder on the lateral portion of the right calf. Participants were instrumented with three microdialysis probes underneath the bladder. One site received lactated ringers (control site), one received the Rho-Kinase inhibitor Fasudil, and one received Ascorbic Acid. Skin temperature (Tskin) and cutaneous vascular conductance (CVC) was measured at each site. Subjects had 1 °C water perfused through the bladder for 30 min, followed by passive rewarming for 90 min. Tskin fell from ~ 34 °C to ~ 18.0 °C during active cooling across all sites and this response was similar for all sites (P>0.05 for all comparisons). During passive rewarming Tskin rose to a similar degree in all sites (P>0.05 relative to the end of cooling). %CVC was reduced during active cooling in all sites; however, the magnitude of this response was blunted in the Fasudil site relative to control (P<0.001 for all comparisons) and min 25 and 30 of cooling in the Ascorbic Acid site (P<0.05). During passive rewarming %CVC at the control and Ascorbic Acid sites did not change such that values were similar to the end of cooling (P>0.05 for each comparison). %CVC at the Fasudil site remained elevated during passive rewarming such that values were higher compared to the control and Ascorbic Acid sites throughout the 90 min of passive rewarming (P<0.001 main effect of Fasudi). These findings indicate that the Rho-kinase pathway contributes to pronounced

  13. Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses

    PubMed Central

    Zeng, Houqing; Xu, Luqin; Singh, Amarjeet; Wang, Huizhong; Du, Liqun; Poovaiah, B. W.

    2015-01-01

    Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes, and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of reactive oxygen species signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of

  14. Neuronal nitric oxide contributes to neuroplasticity-associated protein expression through cGMP, protein kinase G, and extracellular signal-regulated kinase.

    PubMed

    Gallo, Eduardo F; Iadecola, Costantino

    2011-05-11

    Nitric oxide (NO) synthesized by neuronal NO synthase (nNOS) has long been implicated in brain plasticity. However, it is unclear how this short-lived mediator contributes to the long-term molecular changes underlying neuroplasticity, which typically require activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway and gene expression. To address this issue, we used a neuroplasticity model based on treatment of neuronal cultures with bicuculline and a model of experience-dependent plasticity in the barrel cortex. In neuronal cultures, NOS inhibition attenuated the bicuculline-induced activation of ERK and the expression of c-Fos, Egr-1, Arc, and brain-derived neurotrophic factor (BDNF), proteins essential for neuroplasticity. Furthermore, inhibition of the NO target soluble guanylyl cyclase or of the cGMP effector kinase protein kinase G (PKG) reduced both ERK activation and plasticity-related protein expression. NOS inhibition did not affect phosphorylation of cAMP response element-binding protein (CREB), a well-established ERK nuclear target, but it attenuated the nuclear accumulation of the CREB coactivator TORC1 and suppressed the activation of Elk-1, another transcription factor target of ERK. Consistent with these in vitro observations, induction of c-Fos, Egr-1, and BDNF was attenuated in the D1 cortical barrel of nNOS(-/-) mice subjected to single whisker experience. These results establish nNOS-derived NO as a key factor in the expression of proteins involved in neuroplasticity, an effect mediated through cGMP, PKG, and ERK signaling. These actions of NO do not depend on CREB phosphorylation but may involve TORC1 and Elk-1. Our data unveil a previously unrecognized link between neuronal NO and the molecular machinery responsible for the sustained synaptic changes underlying neuroplasticity.

  15. Protein Kinase D Interacts with Neuronal Nitric Oxide Synthase and Phosphorylates the Activatory Residue Serine1412

    PubMed Central

    García-Guerra, Lucía; Pose-Utrilla, Julia; Rodríguez-Crespo, Ignacio; Iglesias, Teresa

    2014-01-01

    Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser916, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser1412, and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions

  16. Protective benefits of AMP-activated protein kinase in hepatic ischemia-reperfusion injury

    PubMed Central

    Zhang, Min; Yang, Dan; Gong, Xianqiong; Ge, Pu; Dai, Jie; Lin, Ling; Zhang, Li

    2017-01-01

    Hepatic ischemia-reperfusion injury (HIRI) is a major cause of hepatic failure and death after liver trauma, haemorrhagic shock, resection surgery and liver transplantation. AMP-activated protein kinase (AMPK) is an energy sensitive kinase that plays crucial roles in the regulation of metabolic homeostasis. In HIRI, ischemia induces the decline of ATP and the increased ratio of AMP/ATP, which promotes the phosphorylation and activation of AMPK. Three AMPK kinases, liver kinase B1 (LKB1), Ca2+/calmodulin-depedent protein kinase kinase β (CaMKKβ) and TGF-β-activated kinase-1 (TAK1), are main upstream kinases for the phosphorylation of AMPK. In addition to the changed AMP/ATP ratio, the activated CaMKKβ by increased intracelluar Ca2+ and the overproduction of reactive oxygen species (ROS) are also involved in the activation of AMPK during HIRI. The activated AMPK might provide protective benefits in HIRI via prevention of energy decline, inhibition of inflammatory response, suppression of hepatocyte apoptosis and attenuation of oxidative stress. Thus, AMPK might become a novel target for the pharmacological intervention of HIRI. PMID:28386315

  17. Regulation of RYR1 activity by Ca(2+) and calmodulin

    NASA Technical Reports Server (NTRS)

    Rodney, G. G.; Williams, B. Y.; Strasburg, G. M.; Beckingham, K.; Hamilton, S. L.

    2000-01-01

    The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.

  18. Regulation of RYR1 activity by Ca(2+) and calmodulin

    NASA Technical Reports Server (NTRS)

    Rodney, G. G.; Williams, B. Y.; Strasburg, G. M.; Beckingham, K.; Hamilton, S. L.

    2000-01-01

    The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.

  19. Possible role of calmodulin in excystation of Giardia lamblia.

    PubMed

    Bernal, R M; Tovar, R; Santos, J I; Muñoz, M L

    1998-09-01

    The protozoan Giardia lamblia initiates infection when trophozoites emerge from a cyst in the hosts by the excystation process. Although this process is crucial to the initiation of infection by G. lamblia, little is known about its regulation. To study the possible involvement of calmodulin (CaM) in excystation we tested the effect of several CaM antagonists (TFP, W-7, and W-5) on this cellular function. Except for W-5 the rest of these compounds inhibited excystation. The protein kinase C inhibitor H-7 had no effect on excystation, suggesting that CaM antagonists acted by selectively inhibiting CaM. Furthermore, CaM was redistributed after the induction of excystation and there was an increase in its fluorescence and activity. These results suggest that a CaM-dependent process is involved in G. lamblia excystation.

  20. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  1. Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells

    PubMed Central

    Barlow, Christy A.; Kitiphongspattana, Kajorn; Siddiqui, Nazli; Roe, Michael W.; Mossman, Brooke T.

    2008-01-01

    Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress. PMID:18392938

  2. Oxidative stress improves coronary endothelial function through activation of the pro-survival kinase AMPK

    PubMed Central

    Liu, Yuhong; Feng, Jun; Cordeiro, Brenda; Lyra, Arthur; Arafah, Mohammed; Yassin-Kassab, Abdulmounem; Zanetti, Arthus V.D.; Clements, Richard T.; Bianchi, Cesario; Benjamin, Laura E.; Sellke, Frank W.; Abid, Ruhul

    2013-01-01

    Age-associated decline in cardiovascular function is believed to occur from the deleterious effects of reactive oxygen species (ROS). However, failure of recent clinical trials using antioxidants in patients with cardiovascular disease, and the recent findings showing paradoxical role for NADPH oxidase-derived ROS in endothelial function challenge this long-held notion against ROS. Here, we examine the effects of endothelium-specific conditional increase in ROS on coronary endothelial function. We have generated a novel binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) that induces endothelial cell (EC)-specific overexpression of Nox2/gp91 (NADPH oxidase) and 1.8±0.42-fold increase in EC-ROS upon tetracycline withdrawal (Tet-OFF). We examined ROS effects on EC signaling and function. First, we demonstrate that endothelium-dependent coronary vasodilation was significantly improved in Tet-OFF Nox2 compared to Tet-ON (control) littermates. Using EC isolated from mouse heart, we show that endogenous ROS increased eNOS activation and nitric oxide (NO) synthesis through activation of the survival kinase AMPK. Coronary vasodilation in Tet-OFF Nox2 animals was CaMKKβ-AMPK-dependent. Finally, we demonstrate that AMPK activation induced autophagy and thus, protected ECs from oxidant-induced cell death. Together, these findings suggest that increased ROS levels, often associated with cardiovascular conditions in advanced age, play a protective role in endothelial homeostasis by inducing AMPK-eNOS axis. PMID:24018842

  3. Calmodulin independence of human duodenal adenylate cyclase.

    PubMed Central

    Smith, J A; Griffin, M; Mireylees, S E; Long, R G

    1991-01-01

    The calmodulin and calcium dependence of human adenylate cyclase from the second part of the duodenum was assessed in washed particulate preparations of biopsy specimens by investigating (a) the concentration dependent effects of free [Ca2+] on enzyme activity, (b) the effects of exogenous calmodulin on enzyme activity in ethylene glycol bis (b-aminoethyl ether)N,N'-tetra-acetic acid (EGTA) washed particulate preparations, and (c) the effects of calmodulin antagonists on enzyme activity. Both basal (IC50 = 193.75 (57.5) nmol/l (mean (SEM)) and NaF stimulated (IC50 = 188.0 (44.0) nmol/l) adenylate cyclase activity was strongly inhibited by free [Ca2+] greater than 90 nmol/l. Free [Ca2+] less than 90 nmol/l had no effect on adenylate cyclase activity. NaF stimulated adenylate cyclase activity was inhibited by 50% at 2.5 mmol/l EGTA. This inhibition could not be reversed by free Ca2+. The addition of exogenous calmodulin to EGTA (5 mmol/l) washed particulate preparations failed to stimulate adenylate cyclase activity. Trifluoperazine and N-(8-aminohexyl)-5-IODO-1-naphthalene-sulphonamide (IODO 8) did not significantly inhibit basal and NaF stimulated adenylate cyclase activity when measured at concentrations of up to 100 mumol/l. These results suggest that human duodenal adenylate cyclase activity is calmodulin independent but is affected by changes in free [Ca2+]. PMID:1752461

  4. A major second messenger mediator of Electrophorus electricus electric tissue is CaM kinase II.

    PubMed

    Gotter, A L; Kaetzel, M A; Dedman, J R

    1997-09-01

    Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca2+ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca(2+)-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca2+/calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca(2+)-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes.

  5. Ablation of Protein Kinase CK2β in Skeletal Muscle Fibers Interferes with Their Oxidative Capacity

    PubMed Central

    Eiber, Nane; Simeone, Luca; Hashemolhosseini, Said

    2017-01-01

    The tetrameric protein kinase CK2 was identified playing a role at neuromuscular junctions by studying CK2β-deficient muscle fibers in mice, and in cultured immortalized C2C12 muscle cells after individual knockdown of CK2α and CK2β subunits. In muscle cells, CK2 activity appeared to be at least required for regular aggregation of nicotinic acetylcholine receptors, which serves as a hallmark for the presence of a postsynaptic apparatus. Here, we set out to determine whether any other feature accompanies CK2β-deficient muscle fibers. Hind limb muscles gastrocnemius, plantaris, and soleus of adult wildtype and CK2β-deficient mice were dissected, cross-sectioned, and stained histochemically by Gomori trichrome and for nicotinamide adenine dinucleotide (NADH) dehydrogenase and succinate dehydrogenase (SDH) enzymatic activities. A reduction of oxidative enzymatic activity was determined for CK2β-deficient muscle fibers in comparison with wildtype controls. Importantly, the CK2β-deficient fibers, muscle fibers that typically exhibit high NADH dehydrogenase and SDH activities, like slow-type fibers, showed a marked reduction in these activities. Altogether, our data indicate additional impairments in the absence of CK2β in skeletal muscle fibers, pointing to an eventual mitochondrial myopathy. PMID:28106831

  6. Differential inhibition of calmodulin-sensitive phosphodiesterase and Ca++-adenosine triphosphatase by chlorpromazine-linked calmodulin

    SciTech Connect

    Prozialeck, W.C.; Wallace, T.L.; Weiss, B.

    1987-10-01

    Upon irradiation with UV light, chlorpromazine binds irreversibly to calmodulin and inactivates it. To determine whether this chlorpromazine-calmodulin (CPZ-CaM) complex can inhibit the actions of native calmodulin, we examined its effects on the activity of calmodulin-sensitive cyclic nucleotide phosphodiesterase from rat brain and on the Ca++-adenosine triphosphatase (ATPase) of human erythrocyte membranes. The CPZ-CaM complex was prepared by irradiating purified bovine brain calmodulin in the presence of chlorpromazine and Ca++. The sample was then dialyzed extensively to remove reversibly bound chlorpromazine and then assayed for its ability to activate calmodulin-sensitive phosphodiesterase and Ca++-ATPase, and for its ability to block the stimulatory effects of native calmodulin on these enzymes. The CPZ-CaM complex had no effect on the basal activity of either enzyme; it neither activated nor inhibited the enzymes when assayed in the absence of calmodulin. However, it affected differentially the activation of the two enzymes by native calmodulin. The CPZ-CaM complex totally inhibited calmodulin-stimulated phosphodiesterase but had no effect on the activation of the ATPase by calmodulin. Other studies showed that CPZ-CaM increased the activation constant (Ka) for the interaction of calmodulin with phosphodiesterase but did not affect the maximal activation (Vmax) of the enzyme by calmodulin. Neither calmodulin nor CPZ-CaM altered the Km for the interaction between phosphodiesterase and cyclic AMP. These results suggest that CPZ-CaM inhibits the calmodulin-induced activation of phosphodiesterase by competing with calmodulin for regulatory sites on the enzyme and not by interacting with calmodulin itself or by blocking the interaction of cyclic AMP with the enzyme.

  7. Mitochondrial and Oxidative Stress-Mediated Activation of Protein Kinase D1 and Its Importance in Pancreatic Cancer

    PubMed Central

    Döppler, Heike; Storz, Peter

    2017-01-01

    Due to alterations in their metabolic activity and decreased mitochondrial efficiency, cancer cells often show increased generation of reactive oxygen species (ROS), but at the same time, to avoid cytotoxic signaling and to facilitate tumorigenic signaling, have mechanism in place that keep ROS in check. This requires signaling molecules that convey increases in oxidative stress to signal to the nucleus to upregulate antioxidant genes. Protein kinase D1 (PKD1), the serine/threonine kinase, is one of these ROS sensors. In this mini-review, we highlight the mechanisms of how PKD1 is activated in response to oxidative stress, so far known downstream effectors, as well as the importance of PKD1-initiated signaling for development and progression of pancreatic cancer. PMID:28361035

  8. Aurintricarboxylic acid protects against cell death caused by lipopolysaccharide in macrophages by decreasing inducible nitric-oxide synthase induction via IkappaB kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase inhibition.

    PubMed

    Tsi, Chin-Ju; Chao, Yee; Chen, Ching-Wen; Lin, Wan Wan

    2002-07-01

    To elucidate the mechanisms involved in cell protection by aurintricarboxylic acid (ATA), an endonuclease inhibitor, high nitric oxide (NO)-induced macrophage apoptosis was studied. In RAW 264.7 macrophages, a high level of NO production accompanied by cell apoptosis was apparent with lipopolysaccharide (LPS) treatment. Direct NO donor sodium nitroprusside (SNP) also dramatically induced cell death, with an EC(50) of 1 mM. Coincubation of ATA (1-500 microM) in LPS-stimulated RAW 264.7 cells resulted in a striking reduction of NO production and cell apoptosis, whereas only a partial cell protection was achieved in response to SNP. This suggests that abrogation of inducible nitric-oxide synthase (iNOS)-dependent NO production might contribute to ATA protection of LPS-treated cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis revealed that ATA down-regulated iNOS protein through transcriptional inhibition of iNOS gene expression but was unrelated to iNOS protein stability. ATA not only inhibited nuclear factor-kappaB (NF-kappaB) activation through impairment of the targeting and degradation of IkappaBs but also reduced LPS-induced activator protein-1 (AP-1) activation. These actions of ATA were not caused by the influence on LPS binding to macrophage membrane. Kinase assays indicated that ATA inhibited IkappaB kinase (IKK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) activity both in vivo and in vitro, suggesting a direct interaction between ATA and these signaling molecules. Taken together, these results provide novel action targets of ATA and indicate that ATA protection of macrophages from LPS-mediated cell death is primarily the result of its inhibition of NO production, which closely relates to the inactivation of NF-kappaB and AP-1 and inhibition of IKK, ERK and p38 MAPK.

  9. Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress.

    PubMed

    Ebrahimian, Talin; Li, Melissa Wei; Lemarié, Catherine A; Simeone, Stefania M C; Pagano, Patrick J; Gaestel, Matthias; Paradis, Pierre; Wassmann, Sven; Schiffrin, Ernesto L

    2011-02-01

    Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation

  10. Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

    PubMed Central

    1985-01-01

    We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole- phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue

  11. Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo.

    PubMed

    Dong, Yunzhou; Zhang, Miao; Wang, Shuangxi; Liang, Bin; Zhao, Zhengxing; Liu, Chao; Wu, Mingyuan; Choi, Hyoung Chul; Lyons, Timothy J; Zou, Ming-Hui

    2010-06-01

    The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo. ER stress markers, sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet. Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5'-aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE(-/-) and ApoE(-/-)/AMPKalpha2(-/-) fed a high-fat diet. We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.

  12. Protein Kinase D1 (PKD1) Phosphorylation Promotes Dopaminergic Neuronal Survival during 6-OHDA-Induced Oxidative Stress

    PubMed Central

    Asaithambi, Arunkumar; Ay, Muhammet; Jin, Huajun; Gosh, Anamitra; Anantharam, Vellareddy; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2014-01-01

    Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD. PMID:24806360

  13. Rho/Rho-kinase pathway in the brainstem contributes to hypertension caused by chronic nitric oxide synthase inhibition.

    PubMed

    Ito, Koji; Hirooka, Yoshitaka; Kishi, Takuya; Kimura, Yoshikuni; Kaibuchi, Kozo; Shimokawa, Hiroaki; Takeshita, Akira

    2004-02-01

    Central nervous system mechanisms are involved in hypertension caused by chronic inhibition of nitric oxide (NO) synthesis. Chronic inhibition of NO synthesis might also activate the Rho/Rho-kinase pathway in the vasculature. We recently demonstrated that activation of the Rho/Rho-kinase pathway in the nucleus tractus solitarii (NTS) contributes to hypertensive mechanisms in spontaneously hypertensive rats. The aim of the present study was to determine whether activation of this pathway also contributes to neurogenic hypertensive mechanisms caused by chronic NO synthesis inhibition. The NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) was administered to Wistar-Kyoto rats in their drinking water (1 mg/mL) for 2 weeks. Bilateral microinjection of Y-27632, a specific Rho-kinase inhibitor, into the NTS elicited decreases in arterial pressure, heart rate, and renal sympathetic nerve activity in control rats and L-NAME-treated rats. The magnitude of the decrease, however, was significantly greater in L-NAME-treated than in control rats. In another group of rats, the specific Rho-kinase inhibitor, Y-27632, was administered intracisternally for 2 weeks with a mini-osmotic pump from the beginning of treatment with L-NAME. Y-27632 co-treatment significantly attenuated the increase in arterial pressure. Furthermore, the expression level of membranous RhoA and phosphorylation of the target proteins of Rho-kinase, the ERM (ezrin, radixin, moesin) family members, was significantly greater in L-NAME-treated rats than in control rats. These results indicate that activation of the Rho/Rho-kinase pathway in the NTS contributes to neurogenic hypertension caused by chronic NO synthase inhibition.

  14. Nitric oxide regulation of gene transcription via soluble guanylate cyclase and type I cGMP-dependent protein kinase.

    PubMed

    Idriss, S D; Gudi, T; Casteel, D E; Kharitonov, V G; Pilz, R B; Boss, G R

    1999-04-02

    Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NON