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1.
Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca2+ signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca2+-dependent electrophoretic mobility shift and Ca2+ binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.  相似文献   

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The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca2+‐ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein‐undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l−1, respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca2+‐ATPase (PMCA) activity, Ca2+/CaM activated EGM Ca2+ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca2+/CaM to activate EGM Ca2+‐ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.  相似文献   

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Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca2+ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca2+ binding to an unidentified secondary Ca2+ sensor. To test whether CaM could be the enigmatic Ca2+ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca2+ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca2+/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.  相似文献   

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Postsynaptic density protein‐95 (PSD‐95) localizes AMPA‐type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca2+ influx, Ca2+/calmodulin (Ca2+/CaM) binding to the N‐terminus of PSD‐95 mediates postsynaptic loss of PSD‐95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD‐95 N‐terminus as important for binding to Ca2+/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaMR126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca2+/CaM to PSD‐95 induced by a chronic increase in Ca2+ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.  相似文献   

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Xu GY  Rocha PS  Wang ML  Xu ML  Cui YC  Li LY  Zhu YX  Xia X 《Planta》2011,234(1):47-59
Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca2+ signals are perceived by different Ca2+ sensors, and calmodulin (CaM) is one of the best-characterized Ca2+ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei’ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca2+ in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.  相似文献   

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Calcium, as the most widely accepted messenger, plays an important role in plant stress responses through calcium-dependent signaling pathways. The calmodulin-like family genes (CMLs) encode Ca2+ sensors and function in signaling transduction in response to environmental stimuli. However, until now, the function of plant CML proteins, especially soybean CMLs, is largely unknown. Here, we isolated a Glycine soja CML protein GsCML27, with four conserved EF-hands domains, and identified it as a calcium-binding protein through far-UV CD spectroscopy. We further found that expression of GsCML27 was induced by bicarbonate, salt and osmotic stresses. Interestingly, ectopic expression of GsCML27 in Arabidopsis enhanced plant tolerance to bicarbonate stress, but decreased the salt and osmotic tolerance during the seed germination and early growth stages. Furthermore, we found that ectopic expression of GsCML27 decreases salt tolerance through modifying both the cellular ionic (Na+, K+) content and the osmotic stress regulation. GsCML27 ectopic expression also decreased the expression levels of osmotic stress-responsive genes. Moreover, we also showed that GsCML27 localized in the whole cell, including cytoplasm, plasma membrane and nucleus in Arabidopsis protoplasts and onion epidermal cells, and displayed high expression in roots and embryos. Together, these data present evidence that GsCML27 as a Ca2+-binding EF-hand protein plays a role in plant responses to bicarbonate, salt and osmotic stresses.  相似文献   

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Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+, CaM exclusively utilizes its C‐terminal lobe (CaMC) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+‐loaded N‐terminal lobe of CaM (CaMN) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+‐free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.  相似文献   

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Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca2+–CaM, Ca2+–CaM–PDE1A, and Ca2+–CaM–MLCK complexes, CPZ apparent dissociation constants (Kds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca2+hCaM M124C–mBBr quenched the fluorescence (Kd = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca2+hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca2+–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca2+–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.  相似文献   

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Plants have evolved numerous receptor-like kinases (RLKs) that modulate environmental stress responses. However, little is known regarding soybean (Glycine max) RLKs. We have previously identified that Glycine soja Ca2+/CAM-binding RLK (GsCBRLK) is involved in salt tolerance. Here, we report that soluble NSF attachment protein receptor proteins BET1s mediate subcellular localization of calmodulin-binding receptor-like cytoplasmic kinases CRCK1s to modulate salt stress responses. Direct interaction between GsCBRLK and GsBET11a was initially identified via yeast two-hybrid and bimolecular fluorescence complementation assays. Further analysis demonstrated conserved interaction between BET1s and CRCK1s. GsCBRLK interacted with all BET1 proteins in wild soybean (Glycine soja) and Arabidopsis, and GsBET11a strongly associated with GsCRCK1a–1d, but slightly with AtCRCK1. In addition, GsBET11a interacted with GsCBRLK via its C-terminal transmembrane domain (TMD), where the entire TMD, not the sequence, was critical for the interaction. Moreover, the N-terminal variable domain (VD) of GsCBRLK was responsible for interacting with GsBET11a, and the intensity of interaction between GsCBRLK/AtCRCK1 and GsBET11a was dependent on VD. Furthermore, GsBET11a was able to mediate the GsCBRLK subcellular localization via direct interaction with VD. Additionally, knockout of AtBET11 or AtBET12 individually did not alter GsCBRLK localization, while GsBET11a expression caused partial internalization of GsCBRLK from the plasma membrane (PM). We further suggest the necessity of GsCBRLK VD for its PM localization via N-terminal truncation assays. Finally, GsBET11a was shown to confer enhanced salt stress tolerance when overexpressed in Arabidopsis and soybean. These results revealed the conserved and direct interaction between BET1s and CRCK1s, and suggested their involvement in salt stress responses.  相似文献   

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Soybean calmodulin isoform 4 (sCaM4) is a plant calcium‐binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo‐) form of sCaM4 possesses a half unfolded structure, with the N‐terminal domain unfolded and the C‐terminal domain folded. This result was unexpected as the apo‐forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high concentrations in cells (0.5–2 mM), we suggest that Mg2+ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg2+ the initially unfolded N‐terminal domain of sCaM4 folds into an α‐helix‐rich structure, similar to the Ca2+ form. We have used the NMR backbone residual dipolar coupling restraints 1DNH, 1DCαHα, and 1DC′Cα to determine the solution structure of the N‐terminal domain of Mg2+‐sCaM4 (Mg2+‐sCaM4‐NT). Compared with the known structure of Ca2+‐sCaM4, the structure of the Mg2+‐sCaM4‐NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg2+‐sCaM4 and CaM‐binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg2+‐sCaM4 does not bind to Ca2+‐CaM target peptides and therefore is functionally similar to apo‐mCaM. The Mg2+‐ and apo‐structures of the sCaM4‐NT provide unique insights into the structure and function of some plant calmodulins in resting cells.  相似文献   

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Although research has extensively illustrated the molecular basis of plant responses to salt and high‐pH stresses, knowledge on carbonate alkaline stress is poor and the specific responsive mechanism remains elusive. We have previously characterized a Glycine soja Ca2+/CAM‐dependent kinase GsCBRLK that could increase salt tolerance. Here, we characterize a methionine sulfoxide reductase (MSR) B protein GsMSRB5a as a GsCBRLK interactor by using Y2H and BiFc assays. Further analyses showed that the N‐terminal variable domain of GsCBRLK contributed to the GsMSRB5a interaction. Y2H assays also revealed the interaction specificity of GsCBRLK with the wild soybean MSRB subfamily proteins, and determined that the BoxI/BoxII‐containing regions within GsMSRBs were responsible for their interaction. Furthermore, we also illustrated that the N‐terminal basic regions in GsMSRBs functioned as transit peptides, which targeted themselves into chloroplasts and thereby prevented their interaction with GsCBRLK. Nevertheless, deletion of these regions allowed them to localize on the plasma membrane (PM) and interact with GsCBRLK. In addition, we also showed that GsMSRB5a and GsCBRLK displayed overlapping tissue expression specificity and coincident expression patterns under carbonate alkaline stress. Phenotypic experiments demonstrated that GsMSRB5a and GsCBRLK overexpression in Arabidopsis enhanced carbonate alkaline stress tolerance. Further investigations elucidated that GsMSRB5a and GsCBRLK inhibited reactive oxygen species (ROS) accumulation by modifying the expression of ROS signaling, biosynthesis and scavenging genes. Summarily, our results demonstrated that GsCBRLK and GsMSRB5a interacted with each other, and activated ROS signaling under carbonate alkaline stress.  相似文献   

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Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP2) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP2 under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca2+ ([Ca2+]cyt) and inhibited the induction of genes involved in Ca2+ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca2+, to affect rice seedling response to osmotic stress.  相似文献   

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It is now generally accepted that non‐genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca2+ is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca2+ increase is ATP‐consuming plasma membrane calcium pump (PMCA). The enzyme is coded by four genes, but isoforms diversity was detected in excitable and non‐excitable cells. It is the only ion pump stimulated directly by calmodulin (CaM). We examined the role of PMCA isoforms composition and CaM effect in regulation of Ca2+ uptake by estradiol, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and their sulfates in a concentration range from 10?9 to 10?6 M, using the membranes from rat cortical synaptosomes, differentiated PC12 cells, and human erythrocytes. In excitable membranes with full set of PMCAs steroids apparently increased Ca2+ uptake, although to a variable extent. In most of the cases, CaM decreased transport by 30–40% below controls. Erythrocyte PMCA was regulated by the steroids somewhat differently than excitable cells. CaM strongly increased the potency for Ca2+ extrusion in membranes incubated with 17‐β‐estradiol and PREG. Our results indicated that steroids may sufficiently control cytoplasmic calcium concentration within physiological and therapeutic range. The response depended on the cell type, PMCA isoforms expression profile, CaM presence, and the steroids structure. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

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