Die Inositolmonophosphatase (IMPase) reguliert als Schlüsselenzym des Phosphatidyl-Inositol-Signalwegs den Abbau des second messenger Inositoltrisphophat (IP3). Als wahrscheinliche molekulare Zielstruktur in der Behandlung der Bipolar Affektiven Störung ist die Erforschung der Regulation der IMPase von hoher medizinischer Relevanz. Seit Jahrzehnten werden Lithiumsalze, die die IMPase in therapeutischen Konzentrationen hemmen, in der Prophylaxe manischer Phasen eingesetzt. Die Therapie mit Lithiumsalzen birgt jedoch die Gefahr zahlreicher schwerwiegender Nebenwirkungen. Außerdem besitzt das Medikament eine geringe therapeutische Breite. Dies macht die Untersuchung alternativer Regulationsmöglichkeiten der IMPase als möglichem Ansatzpunkt für neue pharmakologische Therapien der Bipolar Affektiven Störung hochinteressant. In den letzten Jahren mehrten sich die Hinweise auf eine Regulation der IMPase durch Ca2+-bindende Proteine. Im Rahmen dieser Arbeit wurde mittels verschiedener in vitro-Methoden der Einfluss der Ca2+-bindenen Proteine Calbindin D28k (CB) sowie Calmodulin (CaM) auf die Enzymaktivität der IMPase untersucht. Hierzu wurden die Proteine heterolog in E. coli exprimiert und ein Phosphat-Akkumulationsassay zur Untersuchung der Enzymaktivität der IMPase etabliert. Für die funktionellen Untersuchungen mit CB konnte eine signifikante Steigerung der Aktivität der IMPase nachgewiesen werden. Im Vorfeld der funktionellen Untersuchungen der Enzymaktivität mit CaM konnte eine Ca2+-abhängige Bindung von CaM an die IMPase mittels zweier unabhängiger Experimente gezeigt werden. Da die freie Ca2+-Konzentration einen hemmenden Einfluss auf die Aktivität der IMPase hat, wurde die Ca2+-Bindung durch CaM mit einem Fluoreszenz-Assay kontrolliert. Hierdurch konnten die funktionellen Experimente mit CaM unter genau definierten Ca2+-Konzentrationen durchgeführt werden. In den funktionellen Untersuchungen mit CaM zeigte sich keine Modulation der IMPase. Weiterführende Experimente sollten der Identifizierung möglicher zusätzlicher Interaktionspartner der IMPase dienen. Dazu würden sich Immunpräzipitationsexperimente anbieten. Außerdem sollte eine Untersuchung der Interaktion zwischen IMPase und CaM in einem eukaryotischen Zellsystem erfolgen. Dies würde eine Aussage unter physiologischen Bedingungen ermöglichen.

All organisms require rapid and flexible signaling mechanisms in order to effectively respond to biotic and abiotic stress. Calcium ions (Ca2+) have proven to be important components of signaling networks. Observations of stimulus-specific oscillations of cytosolic Ca2+ during signal transduction suggest that Ca2+ signals directly encode information. These stimulus-specific oscillations, known as Ca2+ signatures, can be interpreted by an array of Ca2+-binding sensors and effectors, which subsequently regulate appropriate cellular responses. While progress has been made regarding the classic Ca2+-sensor calmodulin (CaM), less research has been directed towards the CaM-like family of Ca2+-sensors (CMLs). This family – unique to plants – is suspected to regulate a multitude of stress and developmental pathways; however, to date very few members of this family have had their functions elucidated by the identification of downstream targets and upstream regulators. In the present study, I investigate the regulation of CML39, which has previously been shown to strongly respond to the stress hormone jasmonic acid (JA) in Arabidopsis. Bioinformatic tools predict a large number of putative JA-responsive cis-elements within the CML39 promoter. Deletion analysis of CML39 promoter fragments in planta reveals that some cis-elements respond in a tissue-specific manner. Analysis of transgenic MYC2 loss-of-function (myc2) mutants demonstrates that MYC2 – the preeminent JA-responsive transcription factor – is not necessary for CML39 promoter activity. Collectively, these data reveal a complex tissue-specific pattern of CML39 regulation and provide a foundation for the future identification of relevant transcription factors. / Thesis (Master, Biology) -- Queen's University, 2013-09-24 21:06:30.592

Ceramide-1-phosphate (C1P) has proven to be a bioactive sphingolipid with diverse functions within the cell. At this time, ceramide kinase (CERK) is the only known enzyme known to generate C1P in mammalian cells, and this bioactive lipid is responsible for the activation and translocation of cytosolic phospholipase A2, the initial rate limiting step in eicosanoid synthesis. These studies investigate the regulation of ceramide kinase by calcium. While CERK activity has been shown to be calcium sensitive, little is known about how CERK is activated within the cell; one possibility is the interaction with calcium "sensor" proteins such as calmodulin. In this study, we develop two protocols to efficiently examine the interaction of CERK with calcium dependent proteins: V5 co-immunoprecipitation and Ni-NTA affinity purification. The methods utilize either adenoviral infection or Effectene© transfection of cells to ectopically express CERK with both a 6x His and V5 tag on its C terminus. Unlike the report of Igarashi and co-workers, our findings reveal that CERK does not specifically interact with the calcium sensor, calmodulin, in three different cell types. We also show that the calcium dependent membrane organizer, annexin A2, also does not bind to CERK. In light of these findings, we illustrate that while CERK may be sensitive to calcium, it does not, as previously reported by another laboratory, specifically bind to calmodulin. These studies eliminate possible calcium mediator proteins and are suggestive of another method for the calcium sensitive regulation of CERK lending to new avenues of investigation (i.e. CaMKII). This report also firmly established two successful protocols for investigating protein partners of CERK. Ultimately, through providing a clearer picture behind the calcium regulation of CERK, we can elucidate possible novel therapeutic targets within the inflammatory pathway.

Calcineurin is a Ser/Thr phosphatase whose function is implicated in critical physiological processes such as immune system activation, fetal heart development, and long-term depression in neurons. Calcineurin has been implicated in the progression of Alzheimer’s disease and cardiac hypertrophy. It is not well understood how calcineurin is activated on a molecular level by Ca2+ and its activating protein calmodulin. Previous data from our lab show that calmodulin interaction induces the folding of the intrinsically disordered regulatory domain of calcineurin in two discrete and distant regions into α-helical conformations and that this folding is critical for complete activation of calcineurin. It was also discovered that one of the helical elements which we call the “distal helix” was unstable at a human body temperature of 37°C in dilute buffer. This raises the question; how can a structure critical for the complete activation of calcineurin be unstable at average human body temperature? Proteins do not exist in solutions of the dilute buffer, but rather in a crowded cosmos that ranges between 200 and 400 g/L of macromolecules such as proteins, DNA, and other cellular components. We show here that phenomenon known as macromolecular crowding can stabilize the distal helix and that stabilization increases the activity of calcineurin at human body temperature. Much about intrinsically disordered proteins (IDPs) remains a mystery, especially what influences the rate at which they interact with their target molecules. IDPs lack any sort of stable three-dimensional structure because of their lack of sufficient hydrophobic or aromatic amino acids while having a large proportion of polar and charged amino acids. Because of the high degree of charged amino acids, electrostatic forces play a significant role in their interaction other proteins. This is known to be the case for calmodulin which is net negatively charged protein that has over 300 binding targets of which are usually basic amphipathic alpha-helices. The calmodulin-binding site located in the intrinsically disordered regulatory domain of calcineurin is net positively charged, and, interestingly, is flanked by acidic patches on either side. These acidic patches might perturb attractive electrostatic forces between the calmodulin-binding site and calmodulin. Using fluorescence spectroscopy in conjunction with a stopped-flow apparatus to measure the kinetics between calmodulin and calcineurin we seek to characterize the influence of the steric and electrostatic forces between the two proteins. Also, we present data on RCAN1-4 (Regulator of Calcineurin Isoform 1-4) which has been shown to be an inhibitor in some contexts and an activator of calcineurin in other. RCAN1-4 is expressed in the heart and its upregulation has been shown to prevent calcineurin-mediated pathological cardiac hypertrophy suggesting that it plays an inhibitory role in this context. The work shown demonstrates that RCAN1-4 is a competitive inhibitor of calcineurin and whose binding affinity is modulated by Ca2+/calmodulin. These data unveil a binding site utilized by RCAN1-4 which is commonly used among other calcineurin substrates.

GRK2 wird an Serin29 durch PKC phosphoryliert. Die Phosphorylierung verhindert die Inhibition der GRK2 durch Calmodulin. Die Inhibition der GRK2 durch Calmodulin wird durch den N-Terminus der GRK2 vermittelt und ist auf eine gestörte Aktivierbarkeit der GRK2 durch G-Protein beta/gamma-Untereinheiten zurückzuführen. / GRK2 is phosphorylated by PKC at serin29. The phosphorylation prevents GRK2 inhibition by calmodulin. Inhibition of GRK2 by calmodulin is mediated by the N-terminus of the kinase and is due to a disturbed activation of GRK2 by G-protein beta/gamma subunits.

by Chan Ying Chi, Jessica. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 113-127). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iv / Contents --- p.vi / Abbreviations --- p.xi / Single Letter Amino Acid Code --- p.xvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Glycogen Synthase (EC 2.4.1.11) --- p.1 / Chapter 1.2 --- Glycogen Synthase Kinase-3 --- p.4 / Chapter 1.3 --- Structure of Glycogen Synthase Kinase-3 --- p.5 / Chapter 1.4 --- Functions of Glycogen Synthase Kinase-3 --- p.8 / Chapter 1.4.1 --- Substrate Recognition --- p.8 / Chapter 1.4.2 --- Glycogen Synthase Kinase-3 Homologs --- p.10 / Chapter 1.4.2.1 --- Drosophila --- p.10 / Chapter 1.4.2.2 --- Xenopus --- p.11 / Chapter 1.4.2.3 --- Dictyostelium and Others --- p.12 / Chapter 1.4.3 --- Regulation of Glycogen Synthase-3 in Mammalian Systems --- p.13 / Chapter 1.4.4 --- The role of Glycogen Synthase Kinase-3in Mammalian Brain --- p.16 / Chapter 1.4.4.1 --- Glycogen Synthase Kinase-3β --- p.18 / Chapter 1.4.4.2 --- Glycogen Synthase Kinase-3α --- p.21 / Chapter 1.4.5 --- Glycogen Synthase Kinase-3α in Certain Tumor Cells --- p.23 / Chapter 1.5 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Plasmid Minipreparation --- p.26 / Chapter 2.1.2 --- Large Scale of Plasmid DNA Purification Using QIAGEN-tip500 --- p.28 / Chapter 2.1.3 --- Extraction of Human Blood Genomic DNA --- p.30 / Chapter 2.1.4 --- UV Spectroscopy for determining DNA/RNA Concentration --- p.31 / Chapter 2.1.5 --- Agarose Gel Electrophoresis of DNA --- p.31 / Chapter 2.1.6 --- Purification of DNA Fragment from Agarose Gel using GeneClean III ® (BIO 101 Inc.) Kit --- p.32 / Chapter 2.1.7 --- Restriction Digestion of DNA --- p.32 / Chapter 2.1.8 --- Southern Blot --- p.33 / Chapter 2.1.9 --- Probe Labelling --- p.33 / Chapter 2.1.10 --- Hybridization by Radio-labelling --- p.34 / Chapter 2.1.11 --- DNA Sequencing Reaction --- p.35 / Chapter 2.1.12 --- "Preparation of 6% Polyacrylamide, 8M Urea Denaturing Gel for DNA Sequencing Analysis" --- p.37 / Chapter 2.1.13 --- Preparation of Escherichia coli DH5α Competent Cells --- p.38 / Chapter 2.1.14 --- Modification of 5'Protruding end with T4DNA Polymerase --- p.39 / Chapter 2.1.15 --- Ligation and Transformation of Foregin DNA --- p.39 / Chapter 2.1.16 --- Rapid Screening for the Presence of Plasmid --- p.40 / Chapter 2.2 --- Expression of Glycogen Synthase Kinase-3 / Chapter 2.2.1 --- Preparation of Mammalian cells in Culture --- p.41 / Chapter 2.2.2 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2.3 --- Western Blot Detection of Glycogen Synthase Kianse-3 --- p.43 / Chapter 2.3 --- Assay of Glycogen Synthase Kinase Promoter Activity / Chapter 2.3.1 --- Preparation of SHSY5Y in Culture --- p.45 / Chapter 2.3.2 --- Trypsinization for Removing Adherent Cells --- p.45 / Chapter 2.3.3 --- Transfection of Mammalian Cells by Calcium Phosphate Precipitation --- p.46 / Chapter 2.3.4 --- Stimulation of Transfection Cells by different Chemicals and Preparation of Cell Extract --- p.47 / Chapter 2.3.5 --- CAT-ELISA and β-Gal ELISA Assay --- p.47 / Chapter 2.4 --- Isolation of Glycogen Synthase Kinase-3α 5，Promoter Region / Chapter 2.4.1 --- 5'Rapid Amplification of cDNA End (5'RACE) --- p.48 / Chapter 2.4.2 --- PromoterFinder DNA Walking --- p.49 / Chapter 2.4.3 --- YAC Clone Genomic Construction --- p.50 / Chapter 2.5 --- Construction of Plasmid for Assay of Glycogen Synthase Kinase-3α Promoter Activity --- p.53 / Chapter 2.6 --- Genomic Organization of Glycogen Synthase Kinase-3α --- p.53 / Chapter 2.7 --- Primer Extension Assay / Chapter 2.7.1 --- Isolation of Total RNA by TRIZOL Reagent --- p.57 / Chapter 2.7.2 --- Primer Extension by SuperScript II --- p.57 / Chapter 2.8 --- Reagents and Buffers / Chapter 2.8.1 --- Nucleic Acid Electrophoresis Buffers --- p.59 / Chapter 2.8.2 --- Reagents for Preparation of Plasmid DNA --- p.59 / Chapter 2.8.3 --- Media for Bacterial Culture --- p.60 / Chapter 2.8.4 --- Reagents for Southern Blot --- p.60 / Chapter 2.8.5 --- Reagents for SDS-PAGE --- p.61 / Chapter 2.8.6 --- Reagents for Western Blot --- p.62 / Chapter 2.8.7 --- Reagents for DNA Sequencing --- p.62 / Chapter Chapter 3 --- Isolation of 5´ة Glycogen Synthase Kinase-3α Promoter Region / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.66 / Chapter 3.2.1 --- 5' Rapid Amplification of cDNA End (5'RACE) --- p.66 / Chapter 3.2.2 --- PromoterFinder DNA Walking --- p.68 / Chapter 3.2.3 --- YAC Clone Library Construction --- p.71 / Chapter 3.2.3.1 --- Southern Blotting --- p.71 / Chapter 3.2.3.2 --- Isolation of Sequence Upstream of Glycogen Synthase Kinase-3α region from YAC Clone Using PromoterFider DNA Walking --- p.71 / Chapter 3.2.3.3 --- Sequences of 5，Glycogen Synthase Kinase -3α Promoter --- p.73 / Chapter 3.2.4 --- Primer Extension Assay --- p.78 / Chapter 3.2.5 --- Assay of Glycogen Synthase Kinase-3α Promoter Activity using CAT-ELISA --- p.78 / Chapter 3.2.6 --- Genomic Structure of Glycogen Synthase Kinase-3α --- p.84 / Chapter 3.3 --- Discussion --- p.90 / Chapter 3.3.1 --- Glycogen Synthase Kinase-3a Promoter --- p.90 / Chapter 3.3.2 --- Glycogen Synthase Kianse-3a Promoter Activity --- p.92 / Chapter 3.3.3 --- Prospective and Future Studies --- p.94 / Chapter Chapter 4 --- Expression of Glycogen Synthase Kinase-3 / Chapter 4.1 --- Introduction --- p.96 / Chapter 4.2 --- Results Expression of GSK-3 under Stresses --- p.97 / Chapter 4.3 --- Discussion --- p.105 / Chapter 4.3.1 --- Post-translation regulation of Glycogen Synthase Kinase-3 --- p.105 / Chapter 4.3.2 --- Prospective and Future Studies --- p.107 / Chapter Chapter 5 --- Conclusion --- p.109 / Chapter 5.1 --- Promoter study --- p.110 / Chapter 5.2 --- Genomic organization study --- p.111 / Chapter 5.3 --- Expression study --- p.112 / Reference --- p.113 / Appendices / Appendix I G/C contents of GSK-3α Promoter Region --- p.128 / Appendix II Restriction sites of GSK-3α Promoter Region --- p.134 / Appendix III Primers designed on GSK-3α Promoter Region --- p.139 / Appendix IV Restriction sites of GSK-3α cDNA --- p.142 / Appendix V Vectors --- p.150 / Appendix VI Adaptors Sequences --- p.152 / Appendix VII Anti-GSK-3 Antibody --- p.153 / Appendix VIII Raw data of GSK-3α promoter activity assay --- p.154

"November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 131-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Calmodulin (CaM) is an essential protein found in all eukaryotes ranging from vertebrates to unicellular organisms such as Paramecia. CaM is a calcium sensor protein composed of two domains (N and C) responsible for the regulation of numerous calcium-mediated signaling pathways. Four calcium ions bind to CaM, changing its conformation and determining how it recognizes and regulates its cellular targets. Since the discovery of CaM, most studies have focused on the role of its calcium-saturated form. However, an increasing number of target proteins have been discovered that preferentially bind apo (calcium-depleted) CaM. My study focused on understanding how apo CaM recognizes drugs and protein sequences, and how those interactions differ from those of calcium-saturated CaM. I have used spectroscopic methods to explore CaM binding the drug Trifluoperazine (TFP) and the IQ-motif of the type 2 Voltage-Dependent Sodium Channel (Nav1.2IQp). These studies have shown that both TFP and Nav1.2IQp preferentially bind to the "semi-open" conformation of apo CaM. TFP was shown to be an unusual allosteric effector of calcium binding to CaM. Using 15N-HSQC NMR spectroscopy, I determined the stoichiometry of TFP binding to apo Cam to be 2:1 and to (Ca2+)4-CaM to be 4:1 TFP:CaM. That difference in stoichiometry determined whether TFP decreased or increased the affinity of CaM for calcium. Analysis of residue-specific chemical shift differences indicated that TFP binding to apo and (Ca2+)4-CaM perturbed the C-domain more than the N-domain, prompting high-resolution structural studies of the isolated C-domain of CaM. Crystallographic studies of TFP bound to a calcium-saturated C-domain fragment of CaM (CaM76-148) revealed that CaM adopted an "open" tertiary conformation. The unit cell contained two protein and 4 drug molecules. The orientation of TFP revealed that its trifluoromethyl group was found in two alternative positions (one in each protein in the unit cell), and that Met 144 acted as a gatekeeper to select the orientation of TFP. In contrast to TFP binding to the "open" conformation of calcium-saturated CaM76-148, my NMR studies showed that TFP bound the "semi-open" conformation of apo CaM76-148. TFP interacted with CaM residues near the perimeter of the hydrophobic pocket, but did not contact residues that are solvent-accessible only in the "open" form. Allosteric effects due to TFP binding were observed in the calcium-binding loops of apo CaM76-148. These properties suggest that TFP may antagonize interactions between apo CaM and target proteins such as ion channels that preferentially bind apo CaM. Nav1.2, is responsible for the passage of Na+ ion across cellular membranes. Apo binding of CaM to Nav1.2 poises it for action upon calcium release in the cell. My NMR studies of CaM binding to the Nav1.2 IQ-motif sequence (Nav1.2IQp) showed that the C-domain of apo CaM was necessary and sufficient for binding. My high-resolution structure of the isolated C-domain of CaM bound to Nav1.2IQp revealed that the domain adopted a "semi-open" conformation. At the interface between the IQ-motif and CaM, the highly conserved I and two Y residues of Nav1.2IQp interacted with hydrophobic residues of CaM, while the invariant Q residue interacted with residues in the loop between helices F and G of CaM. This is the first CaM-IQ complex to be determined by NMR; the only other available structure of apo CaM bound to an IQ-motif was determined crystallographically. To accomplish its regulatory roles in response to cellular Ca2+ fluxes, CaM has evolved multiple binding interfaces that are allosterically linked to its Ca2+-ligation state. My studies of CaM binding to TFP and NaV1.2 demonstrate the versatility of CaM functioning as a regulatory protein comprised of domains having separable functions.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 93-108).