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Calmodulin Binding Protein

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Danton H Oday – 1st expert on this subject based on the ideXlab platform

  • cyclin dependent kinase 5 is a Calmodulin Binding Protein that associates with puromycin sensitive aminopeptidase in the nucleus of dictyostelium
    Biochimica et Biophysica Acta, 2013
    Co-Authors: Robert J Huber, Andrew Catalano, Danton H Oday

    Abstract:

    Abstract 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.

  • an extracellular matrix Calmodulin Binding Protein from dictyostelium with egf like repeats that enhance cell motility
    Cellular Signalling, 2011
    Co-Authors: Andres Suarez, Michael A Myre, Robert J Huber, Danton H Oday

    Abstract:

    Abstract CyrA is a novel cysteine-rich Protein with four EGFL repeats that was isolated using the Calmodulin (CaM) Binding overlay technique (CaMBOT), suggesting it is a CaM-Binding Protein (CaMBP). The full-length 63 kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-Binding domain was detected and both CaM-agarose Binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca2+-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM Protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular Protein identified in a lower eukaryote.

  • nucleolar localization and identification of nuclear nucleolar localization signals of the Calmodulin Binding Protein nucleomorphin during growth and mitosis in dictyostelium
    Histochemistry and Cell Biology, 2011
    Co-Authors: Andrew Catalano, Danton H Oday

    Abstract:

    The CalmodulinBinding Protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar Proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and Calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar Protein. Prior to this study, the dynamics of specific nucleolar Proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar Protein and the only nucleolar CalmodulinBinding Protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.

Michael A Myre – 2nd expert on this subject based on the ideXlab platform

  • A NOVEL, ACIDIC, NUCLEAR CalmodulinBinding Protein FROM DICTYOSTELIUM THAT REGULATES NUCLEAR NUMBER*
    , 2020
    Co-Authors: Michael A Myre

    Abstract:

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using 35 S-recombinant Calmodulin (CaM) as a probe has revealed approximately three-dozen Ca 2 dependent Calmodulin Binding Proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel CalmodulinBinding Protein (CaMBP); we have called nucleomorphin, from D. discoideum .A ZAP cDNA expression library of cells from multicellular development was screened using a recombinant Calmodulin probe ( 35 S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca 2 -dependent manner. Amino-terminal fusion to the green fluorescence Protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a CalmodulinBinding Protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. Calmodulin (CaM 1 ), the major, essential Ca 2 –Binding Protein of all eukaryotes, is highly conserved such that the CaM of

  • an extracellular matrix Calmodulin Binding Protein from dictyostelium with egf like repeats that enhance cell motility
    Cellular Signalling, 2011
    Co-Authors: Andres Suarez, Michael A Myre, Robert J Huber, Danton H Oday

    Abstract:

    Abstract CyrA is a novel cysteine-rich Protein with four EGFL repeats that was isolated using the Calmodulin (CaM) Binding overlay technique (CaMBOT), suggesting it is a CaM-Binding Protein (CaMBP). The full-length 63 kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-Binding domain was detected and both CaM-agarose Binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca2+-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM Protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular Protein identified in a lower eukaryote.

  • differentiation inducing factor 1 dif 1 induces gene and Protein expression of the dictyostelium nuclear Calmodulin Binding Protein nucleomorphin
    Cellular Signalling, 2009
    Co-Authors: Danton H Oday, Yekaterina Poloz, Michael A Myre

    Abstract:

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

Yingtang Lu – 3rd expert on this subject based on the ideXlab platform

  • a tobacco calcium Calmodulin Binding Protein kinase functions as a negative regulator of flowering
    Journal of Biological Chemistry, 2004
    Co-Authors: Lei Zhang, Shuping Liang, Russell L Jones, Yingtang Lu

    Abstract:

    Abstract A tobacco calcium/CalmodulinBinding Protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 Protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other Calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco Protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-Binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent Protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.

  • Characterization of a Calmodulin Binding Protein kinase fromArabidopsis thalian
    Chinese Science Bulletin, 2003
    Co-Authors: Ying Wang, Shuping Liang, Yingtang Lu

    Abstract:

    A full-length Calmodulin Binding Protein kinase cDNA,AtCBK1, fromArabidopsis has been isolated by screening of anArabidopsis cDNA library and by 5′-RACE. Northern blot andin situ hybridization indicated that the expression ofAtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal thatAtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests thatAtCBK1 encodes a CaM-Binding serine/threonine Protein kinase.

  • Calmodulin Binding Protein kinases in plants
    Trends in Plant Science, 2003
    Co-Authors: Lei Zhang, Yingtang Lu

    Abstract:

    Many CalmodulinBinding Protein kinases have been isolated from plants. Plant CalmodulinBinding Protein kinases are novel Protein kinases that differ from calcium-dependent Protein kinases in many important respects. CalmodulinBinding Protein kinases are likely to be crucial mediators of responses to diverse endogenous and environmental cues in plants. In this update, we review the structure, regulation, expression and possible functions of plant CalmodulinBinding Protein kinases.