Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases. The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A. Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

Cancer is an ongoing problem all over the world. To find a cure to this disease, both clinicians and scientists are looking for a reasonable treatment method. According to Hanahan and Weinberg, one of the hallmarks of cancer is evasion of programmed cell death, referred to as apoptosis. Apoptosis is an important cellular process, and is regulated by many different pathways. Proteins in these pathways contribute to either cell death or cell survival depending on the cell stresses. Much research in systems biology has been devoted to understanding these pathways at the molecular level. In this study a mathematical model is built to describe apoptosis, and the pathways involving the related proteins p53 and Akt. The primary purpose of the construction of the kinetic model is to verify that this network can exhibit bistability between cell survival and cell death. Sensitivity and bifurcation analysis are conducted to determine which parameters have the greatest effect on the system behavior.

The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX1 and OX2 receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors. Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca2+ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca2+ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX1R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX1R signaling, but also SOCs amplified PLC activity. A technique to block OX1R-induced IP3 increase and subsequent Ca2+ release was devised, leaving ROC influx as the only source of Ca2+ elevation upon OX1R activation. This block had no effect on OX1R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca2+ elevating process in OX1R signaling. OX1Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP3. At even lower orexin-A concentrations, OX1Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.

<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

Cancer is an ongoing problem all over the world. To find a cure to this disease, both clinicians and scientists are looking for a reasonable treatment method. According to Hanahan and Weinberg, one of the hallmarks of cancer is evasion of programmed cell death, referred to as apoptosis. Apoptosis is an important cellular process, and is regulated by many different pathways. Proteins in these pathways contribute to either cell death or cell survival depending on the cell stresses. Much research in systems biology has been devoted to understanding these pathways at the molecular level. In this study a mathematical model is built to describe apoptosis, and the pathways involving the related proteins p53 and Akt. The primary purpose of the construction of the kinetic model is to verify that this network can exhibit bistability between cell survival and cell death. Sensitivity and bifurcation analysis are conducted to determine which parameters have the greatest effect on the system behavior.

The RAS-mitogen-activated protein kinase (MAPK) pathway is an essential signaling mechanism that regulates cellular processes and culminates in the activation of specific gene expression programs. Alterations in the RAS-MAPK signaling cascade can modify epigenetic programs and confer advantages in cell growth and transformation. In fact, deregulation of the cascade is a key event in tumour development with 30% of human cancers harbouring RAS mutations. In breast and pancreatic epithelial cancers, characterization of an aberrant RAS-MAPK pathway has focused on upstream mediators such as receptors and oncogenic RAS molecules but the impact of downstream targets remain poorly defined. Stimulation of the RAS-RAF-MEK-MAPK pathway leads to activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2) which are responsible for the phosphorylation of histone H3 on S10 and S28. We postulate that deregulation of the RAS-MAPK pathway produced by constitutive activation and/ or over-expression of upstream components or mitogen stimulation consequently leads to enhanced MSK1 activity and elevated histone H3 phosphorylation levels. We further hypothesize that MSK1-mediated H3 phosphorylation is critical for immediate early gene (IEG) expression, Ras-driven transformation and is associated with regulatory regions upon gene transcription. In mouse fibroblasts, we present evidence for the critical involvement of MSK1 and H3 phosphorylation as mediators that bridge the aberrant signals driven by the RAS-MAPK pathway with nucleosomal modifications, chromatin remodeling, IEG expression and malignant transformation. We then examined if activation of RAS-MAPK signaling in breast cancer cells elicits similar molecular events. We demonstrate that the RAS-MAPK pathway is induced and enhances the association of MSK1 and H3 phosphorylation on the IEG Trefoil Factor 1 resulting in transcriptional activation. We further observed that mutated K-RAS expression did not correlate with genomic instability or altered signaling in pancreatic cancer cell lines while overexpressed HER2 and EGFR breast cancer cell lines generally exhibit upregulated ERK1/2 and H3 phosphorylation levels. Taken together, our studies contribute to the further understanding of MSK-mediated transcriptional activation in response to RAS-MAPK signaling in oncogene-transformed and cancer cell lines. Inhibition of MSK activity may be an unexplored avenue for combination cancer therapy with abnormal RAS-MAPK signaling pathways.

CD97 gehört zur Familie der Adhäsions-G-Protein gekoppelten Rezeptoren (aGPCR), die aus einem langen extrazellulären N-terminalen Fragment (NTF) und einem nicht-kovalent gekoppelten C-terminalen Fragment (CTF) mit der sieben-transmembranären (TM7) Region und dem intrazellulären Teil bestehen. CD97 wird in malignen Tumoren exprimiert. In der humanen Fibrosarkomzelllinie HT1080 steigert die stabile Überexpression von CD97 die ungerichtete zweidimensionale (2D) Migration einzelner Zellen. Eine Verkürzung von CD97 im CTF auf zwei transmembranäre (TM2) Domänen führt zu einer Suppression der 2D-Migration im Vergleich zu stabil mock-transfektierten HT1080 Kontrollzellen. Wahrscheinlich supprimiert CD97/TM2 die endogene CD97-Wirkung. Unbekannt ist, welche Signalwege durch CD97-Überexpression in HT1080 reguliert werden und welche Signalwege für die Migrationssteigerung von HT1080 verantwortlich sind. Die Klärung dieser Signalwege ist Gegenstand der vorliegenden Arbeit. Die Phosphorylierung von Proteinkinasen ist eine posttranslationale Modifikation zur Regulation der Kinaseaktivität mit nachfolgender Aktivierung oder Inaktivierung eines Signalweges. Daher sind Expression und Phosphorylierung der Proteinkinasen zur Identifikation regulierter Signalwege interessant. Dazu wurden in Lysaten von CD97/TM7, CD97/TM2 und mock-transfektierten HT1080 mittels Kinetworks Phosphosite Screen KPSS 1.3 Profiling (Multi-Immunoblot™) 37 verschiedene Proteinphosphorylierungen untersucht und regulierte Signalwege identifiziert. An 25 Phosphorylierungsstellen erfolgt eine Regulation durch CD97. Anschließend wurden die Ergebnisse der interessantesten Proteine hinsichtlich ihrer Expression und Phosphorylierung im Western Blot verifiziert und um Proteine erweitert, die klassisch an der Regulation der Zellmigration beteiligt sind. Es zeigt sich eine Aktivierung des PI3-Kinase/Akt-Signalweges und eine Inhibierung von Src durch CD97. 2D-Migrationsversuche von HT1080 CD97/TM7, CD97/TM2 und mock mit spezifischen Inhibitoren gegen den PI3-Kinase/Akt-Signalweg und gegen Src bestätigen, dass diese Kinasen an der CD97-induzierten Steigerung der 2D-Migration beteiligt sind. Weiterhin finden sich Hinweise, dass in HT1080 CD97 die Apoptose hemmt und die Proliferation reguliert. Insgesamt wird in dieser Arbeit ein Überblick über die durch CD97 regulierten Signalwege gegeben. Die CD97-gesteigerte 2D-Migration von HT1080 wird durch eine Aktivierung des PI3-Kinase/ Akt-Signalweges und Inhibierung von Src vermittelt.

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.