Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.

Calmodulin is an essential EF-hand protein with a helix-loop-helix calcium binding motif. Understanding Ca(II) dependent activation of calmodulin and other EF-hand proteins is limited by Ca(II)-induced conformational change, multiple and cooperative binding of Ca(II) ions, and interactions between the paired EF-hand motifs. The goal of this research project is to probe key determinants for calcium binding properties and pairing interactions at the site specific level using a grafting approach and high resolution NMR. An individual Ca(II) binding site of the EF-hand motifs of calmodulin was grafted into a non-calcium dependent protein, CD2, to bypass limitations associated with natural EF-hand proteins and peptide fragments. Using high resolution NMR, we have shown that the grafted EF-loop III of calmodulin in the host protein retains its native conformation with a strong loop and β-conformation preference. Grafted ligand residues in the engineered protein are directly involved in binding of Ca(II) and La(III). The NMR studies support our hypothesis that both ligand arrangement and dynamic properties play essential role in tuning Ca(II) binding affinities. Using pulse-field diffusion NMR and protein engineering, we further demonstrated that grafted EF- loop remains as a monomer. Although the EF-loop with flanking helices dimerizes in the presence of Ca(II). Additionally, removal of conserved hydrophobic residues at the flanking helices of the EF-hand motif leads to be monomer in the absence and presence of metal ions. Our results suggest that conserved hydrophobic residues are essential for the pair-paired interaction in the coupled EF-hand protein. We have shown that our developed grafting approach can be applied to probe intrinsic Ca(II) binding affinities of different Ca(II) binding sites.

Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.

The increase of calcium in the cell can induce cellular functions such as fertilization, cell division and cell communication. Calcium (Ca2+) carries out these processes through proteins called calcium sensors. An important calcium modulator is calmodulin. Calmodulin has four possible Ca2+ binding sites that have the characteristic helix-loop-helix (EF hand) motif. When the EF hands bind to Ca2+, methionine rich hydrophobic patches are exposed allowing for CaM to interact with target proteins. However, there are proteins that can interact with CaM at low levels of Ca2+ or in the absence of Ca2+. An enzyme that is activated by CaM is nitric oxide synthase (NOS), which converts L-arginine to L-citrulline and nitric oxide (•NO), where •NO is used to carry out important cellular functions. There are three isoforms of the enzyme; endothelial, neuronal and inducible NOS. The first two isoforms are activated by Ca2+-bound CaM when there is an influx of Ca2+ and are therefore Ca2+-dependent whereas inducible NOS (iNOS) is activated and binds tightly to CaM regardless of the Ca2+ concentration and is therefore Ca2+-independent. Of particular interest is the iNOS enzyme, since no three-dimensional structures of the reductase domain or the CaM-binding region have been solved. All three isoforms of NOS exist as homodimers, where each monomer consisting of a reductase domain and an oxygenase domain separated by a CaM-binding region. The reductase domain contains binding sites for NADPH and the flavins, FAD and FMN, which facilitate electron transfer from the NADPH to the catalytic heme in the oxygenase domain of the opposite monomer. The transfer of electrons from the FAD to the heme is carried out by the FMN domain which is proposed to swing between the two docking points since the distance between the two points is too large for electron transfer. This electron transfer point is under the control of CaM, which is essential for NOS activation. This dynamic process and the direct role of CaM have yet to be observed structurally. A method to monitor dynamics structurally is through the use of nuclear magnetic resonance (NMR) spectroscopy. Therefore as the first step to determine the NMR structure of the FMN domain with the CaM-binding region, the structure of the iNOS CaM-binding region bound to CaM will be determined. The structure will allow for further characterization and identification of important interactions between the iNOS CaM-binding region and CaM which contribute to the unique properties of iNOS.

Calcium and calmodulin are implicated in mediating the Ca2+-dependent regulation of gap junctions that are essential for the intercellular transmission of molecules such as nutrients, metabolites, metal ions and signal messengers (< 1000 Da) through its specialized cell membrane channels and communication to extracellular environment. To understand the key determinants for calcium and calmodulin regulation of gap junction, in this study, we identified a calmodulin binding domain in the second half of the intracellular loop of Cxonnexin50 (the major gap junction protein found in an eye lens) using peptide fragments that encompass predicted CaM binding sites and various biophysical methods. Our study provides the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx50 in a Ca2+-dependent manner. Furthermore, two novel CaM binding regions in cytosolic loop and C-termini of Connexin43 (the most ubiquitous connexin) have been shown to interact with CaM with different binding modes in the presence of Ca2+ using high resolution NMR. Our results also elucidate the molecular determinants of regulation of gap junction by multiple CaM targeting regions and provide insight into the molecular basis of gap junction gating mechanism and the binding of CaM to the cytoslic region Cx43-3p as the major regulation site. Upon response to the cytosolic calcium increase, CaM binds to the cytosolic loop to result in the conformational change of gap junction and close the channel. It is possible for CaM to use an adjacent region as an anchor close to the regulation site to allow for fast response. Since a large number of residues in the Cxs mutated in human diseases reside at the highly identified CaM binding sites in Cxs, our studies provide insights into define the critical cellular changes and molecular mechanisms contributing to human disease pathogenesis as part of an integrated molecular model for the calcium regulation of GJs. In addition, we have applied the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor (Shr) of Streptococcus pyrogenes as well as the nonstructural protein 1 (nsP1) of Sindbis virus and Poxvirus. This fast and robust method allows us to analyze putative EF-hand proteins at genome-wide scale and to further visualize the evolutionary scenario of the EF-hand protein family. Further, mass spectrometry has also been applied to probe modification of proteins such as CaM labeling by florescence dye and 7E15 by PEG.

The increase of calcium in the cell can induce cellular functions such as fertilization, cell division and cell communication. Calcium (Ca2+) carries out these processes through proteins called calcium sensors. An important calcium modulator is calmodulin. Calmodulin has four possible Ca2+ binding sites that have the characteristic helix-loop-helix (EF hand) motif. When the EF hands bind to Ca2+, methionine rich hydrophobic patches are exposed allowing for CaM to interact with target proteins. However, there are proteins that can interact with CaM at low levels of Ca2+ or in the absence of Ca2+. An enzyme that is activated by CaM is nitric oxide synthase (NOS), which converts L-arginine to L-citrulline and nitric oxide (•NO), where •NO is used to carry out important cellular functions. There are three isoforms of the enzyme; endothelial, neuronal and inducible NOS. The first two isoforms are activated by Ca2+-bound CaM when there is an influx of Ca2+ and are therefore Ca2+-dependent whereas inducible NOS (iNOS) is activated and binds tightly to CaM regardless of the Ca2+ concentration and is therefore Ca2+-independent. Of particular interest is the iNOS enzyme, since no three-dimensional structures of the reductase domain or the CaM-binding region have been solved. All three isoforms of NOS exist as homodimers, where each monomer consisting of a reductase domain and an oxygenase domain separated by a CaM-binding region. The reductase domain contains binding sites for NADPH and the flavins, FAD and FMN, which facilitate electron transfer from the NADPH to the catalytic heme in the oxygenase domain of the opposite monomer. The transfer of electrons from the FAD to the heme is carried out by the FMN domain which is proposed to swing between the two docking points since the distance between the two points is too large for electron transfer. This electron transfer point is under the control of CaM, which is essential for NOS activation. This dynamic process and the direct role of CaM have yet to be observed structurally. A method to monitor dynamics structurally is through the use of nuclear magnetic resonance (NMR) spectroscopy. Therefore as the first step to determine the NMR structure of the FMN domain with the CaM-binding region, the structure of the iNOS CaM-binding region bound to CaM will be determined. The structure will allow for further characterization and identification of important interactions between the iNOS CaM-binding region and CaM which contribute to the unique properties of iNOS.

<p>Ca<super>2+</super>/Calmodulin-dependent protein kinase kinase &beta;(CaMKK&beta;) is a serine/threonine directed kinase which is activated following increases in intracellular Ca<super>2+</super>. CaMKK&beta; activates Ca<super>2+</super>/Calmodulin-dependent protein kinase I (CaMKI), Ca<super>2+</super>/Calmodulin-dependent protein kinase IV (CaMKIV), and the AMP-dependent protein kinase (AMPK) in a number of physiological pathways including learning and memory formation, neuronal differentiation, and regulation of energy balance. The purpose of the work presented in this dissertation is to better understand the regulation of CaMKK&beta; activity and specificity in CaMKK&beta;-dependent signaling cascades. First, the CaMKK&beta;-AMPK signaling complex is examined using biochemical assays. In both brain and cell lysates CaMKK&beta; and AMPK form a stable complex which can be examined by co-immunoprecipitation. This complex lacks the AMPK&gamma; subunit and is not allosterically activated by adenosine 5'-monophohphate (AMP) binding. Using a series of CaMKK&beta; and AMPK mutants it was determined that the kinase domains of CaMKK&beta; and AMPK are necessary for their interaction and CaMKK&beta; must be active and bound to adenosine 5'-triphosphate (ATP) to form a complex with AMPK. However, CaMKK&beta; need not be active or bound to ATP to bind CaMKIV. This illustrates that the CaMKK&beta;-AMPK signaling complex differs from the CaMKK&beta;-CaMKIV signaling complex. These observations indicate that the CaMKK&beta;-AMPK signaling complex could be specifically targeted without effecting CaMKK&beta;-CaMKIV signaling.</p><p>Second, the regulation of CaMKK&beta; by multi-site phosphorylation is examined. Three phosphorylation sites in the N-terminus of CaMKK&beta; were identified by mass spectrometry which regulates its Ca<super>2+</super>/CaM-independent autonomous activity. The kinases responsible for these phosphorylations are identified as CDK5 and GSK3. These phosphorylation events are sequential with CDK5 priming for subsequent GSK3 phosphorylation. In addition to regulation of autonomous activity, phosphorylation of CaMKK&beta; regulates its half-life as determined in a radioactive pulse-chase assay. Examination of CaMKK&beta; in a cerebellar granule neuron model system demonstrates that CaMKK&beta; levels correlate with CDK5 activity and are regulated developmentally. In addition, appropriate phosphorylation of CaMKK&beta; is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKK&beta;-dependent signaling cascades. </p><p>Overall the work presented in this dissertation illustrates additional levels of regulation of CaMKK&beta;-dependent signaling pathways. In the future, these novel methods of CaMKK&beta; regulation will need to be considered when studying CaMKK&beta;-dependent signaling pathways.</p> / Dissertation

Salvia miltiorrhiza (SM) preconditioning was reported to be helpful in the early recovery of gastrointestinal motility in the intestinal congestion of rats with hepatic ischemia reperfusion. The aim of this study was to determine whether SM stimulates contraction of isolated terminal ileum of Sprague-Dawley rat ex vitro and the mechanisms which regulates that. The roots of SM were extracted by ethanol. One of the indicative marker of SM, Tanshinone IIA, was identified and quantified with high performance liquid chromatography (HPLC), and the results showed that Tanshinone IIA was 1190 £gg/ml in SM extract. The effects of contractile activity of SM extract at various cumulative dosages on the rat isolated terminal ileum were studied in organ bath. The area under curve above the baseline of contractile graphy of SM extract on isolated terminal ileum was recorded. In order to explore the contractile mechanism of SM extract on isolated terminal ileum, the individual pretreatment or use of atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), Ca2+ free Kreb¡¦s solution with EGTA, or trifluoperazine (a calmodulin blocker) was given and then cumulative dosages (40 £gL, 100 £gL, 180 £gL, 280 £gL) of SM extract were added. In addition, we used Fura-2 pentakis acetoxymethyl ester to detect the change of intracellular calcium concentration of intestinal epithelial cell-6 (IEC-6) induced in 1/1000-time or 1/10000-time dilution of SM extract. The result indicated SM extract significantly simulated the contraction of isolated terminal ileum in a dose-dependent manner. The individual addition or use of atropine, tetrodotoxin, nifedipine, or Ca2+ free Kreb¡¦s solution with EGTA all could not down-regulate significantly the contraction of SM extract on isolated terminal ileum. Trifluoperazine significantly down-regulated the contraction of SM extract on isolated terminal ileum. In addiation, SM extract was able to increase cytosolic calcium concentration of IEC-6 cells. In conclusion, the mechanisms of contraction of SM extract on isolated terminal ileum of rat were involved in calmodulin/Ca2+ associated contraction pathway.

Ethylene is a gaseous growth regulator, and plays an important role in response to plant developmental and environmental stimuli. Ethylene also plays a key role in leaf senescence. Calcium is a second message, and participates in the signal transduction pathways of many plant physiological responses. In this research, ethephon, an ethylene-releasing compound, was used to induce sweet potato leaf yellowing, chlorophyll content reduction, photochemical Fv/Fm decrease, H2O2 elevation and senescence-associated gene expression. These ethephon-mediated effects were all delayed or repressed by pretreatment of a calcium ion chelator, EGTA. Treatment with a calcium ionophore A23187 also induced senescence-associated gene expression in sweet potato detached leaves, and the induction was repressed by EGTA pretreatment. Calcium signaling in general is transmitted by calcium sensor proteins, including calmodulin to translate into appropriate responses to developmental and environmental stimuli. Therefore, pretreatment with calmodulin inhibitor chlorpromazine (CPZ) delayed or repressed ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression. These CPZ-mediated effects were reversed by the exogenous application of an ethephon-inducible calmodulin SPCAM fusion protein. These results suggest that external Ca2+ influx and calmodulin SPCAM play a role in ethephon signaling leading to leaf senescence, H2O2 elevation and senescence-associated gene expression. In addition, NaCl salt stress also caused sweet potato leaf senescence, H2O2 elevation and senescence-associated gene expression. Pretreatment with CPZ delayed or repressed NaCl salt stress-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression. These CPZ-mediated effects were also reversed by the exogenous application of calmodulin SPCAM fusion protein. These results suggest that calmodulin SPCAM may play a role in NaCl salt stress signaling leading to leaf senescence, H2O2 elevation and senescence-associated gene expression. Based on these results, external Ca2+ influx is required for ethephon induced leaf senescence. Ethephon-inducible calmodulin SPCAM likely participates in ethylene and NaCl salt stress signaling leading to leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato in order to cope with different developmental cues or environmental stimuli.

Calmodulin (CaM) is a highly conserved protein able to bind Ca2+. When Ca2+ is bound the protein can bind and activate further proteins with several individual functions. CaM switches to a more open conformation when Ca2+-bound and is able to do so at a high rate. Little is known about the conformational switches between apo and Ca2+-bound states. A hypothesis suggests that protonation/deprotonation of a histidine side-chain is part of the answer and thus the dynamics of CaM would be pH dependent. This was further investigated in this thesis. Methods to carry out the project included protein expression of isotope labelled CaM-TR2C E140Q, standard protein purification and protein adapted Nuclear Magnetic Resonance (NMR) spectroscopy. The results suggest that CaM-TR2C E140Q is likely to depend on pH and that histidine 107 (H107) may have a central role in the conformational changes observed. At lower pH it was also suggested that CaM-TR2C E140Q obtained a more open conformation with weakened intramolecular interactions and that the tertiary structure of CaM-TR2C E140Q may have been disrupted. / Calmodulin (CaM) är ett, till hög grad konserverat protein med möjlighet att binda in Ca2+. Då Ca2+ är bundet kan proteinet binda och aktivera ytterligare protein med olika enskilda funktioner. CaM byter med hög hastighet till en mer öppen konformation då Ca2+ binder. Lite vetskap finns kring hur konformationsändringarna mellan apo-form och Ca2+-bunden form går till. En hypotes föreslår att protonering/deprotonering av en histidin-sidokedja kan vara en del av svaret och att CaMs dynamik därför bör vara beroende av pH. Detta undersöktes vidare i detta examensarbete. Metoder som användes för att genomföra projektet inkluderar proteinuttryck av isotopinmärkt CaM-TR2C E140Q, standardiserad proteinrening och proteinanpassad kärnmagnetisk resonans (NMR) spektroskopi. Resultaten föreslår att konformationsändringarna av CaM-TR2C E140Q troligen är pH-beroende och att histidin 107 (H107) kan ha en central roll vid dessa ändringar. Vid lägre pH föreslås att CaM-TR2C E140Q antar en mer öppen konformation med försvagade intramolekylära interaktioner och att tertiärstrukturen av CaM-TR2C E140Q kan ha blivit upplöst.