Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter. Keywords: strain_or_line_design

Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter.

Project description:BACKGROUND:Sustained ?-adrenergic receptor (?-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. OBJECTIVE:The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained ?-AR stimulation and associated functional implications on IKs. METHODS:KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained ?-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral ?CaMKII overexpression. RESULTS:Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained ?-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest ?CaMKII phosphorylation. Sustained ?-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained ?-AR). Individual phosphorylated mimics at S484 (P = .04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). ?CaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). CONCLUSION:CaMKII regulates KCNQ1 at S484 during sustained ?-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

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Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32.

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Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32.

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