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.

Cells transduce signals from the external environment to the inside through phosphatidylinositol-3,4,5-phosphate (PIP3), a major signaling lipid on the plasma membrane. PIP3 is generated by the action of a family of lipid kinases called Class I phosphoinositide 3-kinases (PI3Ks) and controls an array of essential cellular functions including growth, proliferation, survival, metabolism and cytoskeletal architecture. PI3Ks are large heterodimeric complexes composed of a catalytic p110 subunit and a regulatory subunit. Crucial to healthy PIP3 production is the interpretation of diverse activating inputs arising from signaling proteins on the membrane by these subunits. A member of the PI3K family, PI3Kγ is a master regulator of immune functions with therapeutic implications in cancer immunity and inflammatory disease. PI3Kγ is distinct from other well studied PI3Ks due to the presence of unique regulatory mechanisms that control its ability to integrate signals from G-protein coupled receptors, small GTPases, immunoglobulin receptors and toll-like receptors. However, unlike the other well characterized PI3Ks, there are significant gaps in understanding of the molecular details of these mechanisms and how regulatory processes are translated into functions elicited by PI3Kγ in its unique milieu within the immune system. To understand PI3Kγ regulation, I utilized a synergy of cutting-edge approaches including protein biochemistry, X-ray crystallography, cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry to elucidate the unique regulatory features within its catalytic and regulatory subunits and how these features are disrupted in disease. These studies significantly advanced our understanding of how this enzyme functions and provided novel avenues for potentially targeting the enzyme better in therapy. This dissertation will consist of an introduction chapter summarizing PI3Kγ regulation and its role in disease, followed by three data chapters investigating previously uncharacterized regulatory mechanisms that control its function and how these mechanisms are implicated in disease. These data chapters are followed by a final chapter describing conclusions and future directions. In summary, the work presented in this thesis provides novel insights into the unique regulatory features in the catalytic and regulatory subunits of PI3Kγ that mediate its stimulation by upstream activating partners and the mechanisms by which these features are disrupted in disease. Further, these studies have facilitated the effective characterization of potent molecules that can specifically target PI3Kγ in disease. Altogether, the findings of this dissertation constitute a major advancement in our understanding of PI3K regulation. / Graduate

Phytochemicals are recognized as playing an important role in cancer prevention by fruits and vegetables. The avocado is a widely grown and consumed fruit that is high in nutrients and low in calories, sodium, and fats. Studies have shown that phytochemicals extracted from the avocado fruit selectively induce cell cycle arrest, inhibit growth, and induce apoptosis in precancerous and cancer cell lines. Our recent studies indicate that phytochemicals extracted with chloroform from avocado fruits target multiple signaling pathways and increase intracellular reactive oxygen leading to apoptosis. This review summarizes the reported phytochemicals in avocado fruit and discusses their molecular mechanisms and targets. These studies suggest that individual and combinations of phytochemicals from the avocado fruit may offer an advantageous dietary strategy in cancer prevention.

Most existing studies of receptor signaling are qualitative, which can lead scientists to misinterpret or overlook key information about the extent and timing of key events. To overcome these shortcomings, we have applied quantitative approaches to characterize receptor activation and signaling events. Most signaling studies focus on events occurring at a particular level in the system (e.g., on the membrane, at the level of phosphorylation of intracellular signaling molecules, or at the level of transcription). Instead, we are interested in taking a longitudinal view of signaling by achieving a quantitative understanding of a single signaling pathway from initial stimulation of the receptor by its growth factor (GF) ligand, through to gene expression, and functional cellular responses. As a model system for our studies, we used the growth factor receptor tyrosine kinase, REarranged during Transfection (RET), which requires a ligand and a glycosylphosphatidylinositol-anchored co-receptor for activation. RET mediates the response of cells to members of the glial cell-line derived neurotrophic factor (GDNF) family of neurotrophins, which are important in the development and maintenance of a subset of neuronal cells as well as in other cell types and tissues. We have characterized the molecular mechanisms of RET activation and signaling by pursuing the following four aims: 1) We developed a sensitive and robust luciferase reporter gene assay for RET signaling. 2) We characterized the dynamic relationship between receptor activation and downstream signaling events, including gene transcription and translation of three target genes. 3) We used the reporter gene assay, and other detection approaches, to test and quantify crosstalk between RET and other GF receptors. 4) We developed a FRET reporter system to enable monitoring of the assembly of the activated RET receptor complex on cells, as a means to distinguish between ligand-induced oligomerization and pre-associated oligomer mechanisms. Through these four aims, we have established new methods to quantitatively elucidate mechanisms of GF receptor activation, new insights into how signals are propagated from the receptor to the nucleus and into a functional response, and have established crosstalk between RET and other GF receptor pathways.

Leydig cells produce testosterone in response to luteinizing hormone (LH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A pathway. Additionally, these cells are responsible for producing insulin-like peptide 3 (INSL3), a peptide hormone that is essential for testicular descent. The insl3 promoter in Leydig cells can be activated by cAMP through the transcription factor Nur77, which has also been shown to regulate the promoters of the steroidogenic enzymes, cyp17 and 3b-hsd. While the mechanism of LH action on testosterone production is well characterized, the effect of LH on insl3 abundance has yet to be shown directly. The MA-10 Leydig cells treated with hCG exhibited a transient and robust increase in nur77 mRNA, while insl3 mRNA abundance remained unchanged. Further, cAMP failed to affect insl3 mRNA, though nur77 mRNA abundance was significantly increased. Inhibition of LH-receptor-linked signal transduction pathways in the presence of hCG implicated multiple signaling networks in the regulation of both insl3 and nur77. Treatment with hCG or cAMP did not affect the abundance of 3b-hsd mRNA. Interestingly, though the MA-10 cell line has been reported to lack CYP17 activity and mRNA and so produce progesterone instead of testosterone, cyp17 mRNA was present and inducible by hCG and cAMP. The addition of hCG, testosterone, nor the combination of hCG and testosterone affected insl3 mRNA abundance. Though hCG consistently increased nur77 mRNA abundance, the addition of testosterone did not enhance the effects of hCG. Collectively, these results indicate that insl3 is regulated by factors other than LH/CG and cAMP in the MA-10 cell line.

Vascular endothelial growth factor (VEGF) is required for the development, growth and survival of blood vessels. Endothelial cell behavior is altered by cell substrate stiffness, suggesting that VEGF activity might also be influenced by cell-substrate mechanics. We studied VEGF binding, internalization, and signaling as a function of substrate stiffness using endothelial cells cultured on fibronectin (fn) linked polyacrylamide gels. Individual cell analysis of VEGF-induced calcium fluxes in endothelial cells on various stiffness extracellular matrices (ECM) revealed heterogeneity in our cell population that would have been lost using population based averaging. Cluster analysis of individual cells identified two key groups of reacting cells- a minor fraction of highly reactive cells and the bulk of the cells with minimal activation. At subsaturating VEGF doses, highly active cells were phenotypically smaller and thinner than the bulk population. Overall, cells on our softest substrates (4 kPa) were most sensitive to VEGF. To better understand the mechanisms underlying the changes in VEGF signaling due to stiffness, we explored how matrix binding of VEGF and tethering of cells to the matrix modulates VEGF processing. VEGF-ECM binding was enhanced with heparin pre-treatment, which exposed a cryptic VEGF binding site in the fn ECM. Cell produced ECM on the softest substrates were least responsive to heparin, but the cells internalized more VEGF and showed enhanced VEGF signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF in all conditions. β1 integrin, which connects cells to fn, modulated VEGF uptake in a stiffness dependent fashion. β1 protein levels were consistent with stiffness, yet cells on hard surfaces showed greater decreases in VEGF internalization than cells on softer matrices after β1 inhibition. Stiff matrices facilitate the unfolding of fn, which may reduce the binding capacity of β1 integrin. Thus a greater proportion of activated β1 integrin may be sensitive to inhibition in the stiff condition as compared to the soft. Ultimately, through analysis of individual and population-based VEGF-cell responses to stiffness, this study provides insight into how signaling dynamics, cell heterogeneity, and microenvironment influence tissue regeneration and response to injury and disease.

The migration of the cranial neural crest is an essential part of cranio-facial development in every vertebrate embryo. The cranial neural crest (CNC) is a transient population of cells that forms the lateral border of the anterior neural plate. In the tailbud stage Xenopus embryo, the neural crest cells delaminate from the neural tube, and undergo a large-scale migration from the dorsal to ventral region of the embryo. The CNC travels along distinct pathways, and populates specific regions of the embryos face. Once the CNC ceases migrating, it differentiates into a variety of tissues that are essential for cranio-facial structure and function. Some of these tissues include bones, muscle, cartilage, and ganglia. The CNC receives a concert of signals from neighboring tissues during and after CNC migration as well as signals transmitted among CNC cells, which act together to determine the fate of each CNC cell. Therefore, the proper migration of the CNC is an essential part of cranio-facial development. What molecules are important for the process of CNC migration? As one might imagine, a milieu of different molecules and interactions are essential for this complicated embryological process to occur. The work presented in this dissertation will focus on the role of a cell adhesion molecule that is important for Xenopus CNC migration. Typically, the amount of cell adhesion decreases within tissues undergoing migration. This behavior is essential to allow fluidity within the tissue as it moves. However, cell adhesions are fundamental for cell migration to occur because the moving cells need a platform on which to mechanically propel themselves. These interactions can occur between the migrating cell and extracellular matrix molecules (ECM), or can happen between cells. The cranial neural crest utilizes both cell-ECM and cell-cell interactions during the process of migration. The amount of cell adhesion mediated by either of these mechanisms will depend on where the cell is located within the CNC. Cells located at the periphery of the CNC tissue, which is surrounded by a matrix of ECM, will have more cell-ECM interactions. Cells located deeper in the CNC tissue, where there is little ECM, will rely more on cell-cell interactions. The work presented in this thesis focuses on a cell-cell adhesion molecule that is part of the cadherin superfamily of molecules. With this in mind, these studies should be descriptive of the environment within the CNC, and to a less degree the environment between the CNC and the surrounding tissues. The work presented in this dissertation will focus on cadherin-11, which is a classical cadherin that is specifically expressed in the cranial neural crest during its migration. How does cadherin-11 function in the CNC during this process? The work presented here suggests that the main role of cadherin-11 in the CNC is to perform as a cell adhesion molecule. However, too much cell adhesion is inhibitory to migration. In this respect, many of the studies described in this work indicate that cadherin-11 mediated cell adhesion is tightly regulated during CNC migration. Here I show that cadherin-11 is extracellularly processed by ADAM metalloproteases, ADAM9 and ADAM13, which removes the adhesive domain of cadherin-11. This extracellular cleavage event occurs throughout CNC migration, and is likely the main mechanism that regulates cadherin-11 mediated cell adhesion. Cleavage of cadherin-11 by ADAMs does not seem to affect its ability to interact with cytoplasmic binding partners, â-catenin and p120-catenin. This observation supports the idea that the “purpose” of cadherin-11 cleavage is to regulate cell adhesion, and not to induce (cell autonomous) signaling events. Additionally, the secreted extracellular domain of cadherin-11 (EC1-3) retains biological activity. This fragment can bind to a number of cell surface molecules in tissue culture including full-length cadherin-11 and specific members of the ADAM family. This observation suggests that EC1-3 may interact with full-length cadherin-11 molecules in vivo, and inhibit cadherin-11 mediated cell adhesion during CNC migration. EC1-3 can rescue CNC migration in embryos that overexpress cadherin-11, further supporting this hypothesis. Many of the above observations have been published in my first-author paper entitled “Extracellular processing of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest migration” published in the journal Molecular Biology of the Cell in 2009. Some of the unpublished work in this dissertation further focuses on how EC1-3 effects CNC migration in an ex vivo environment. During these studies, the observation was made that overexpression of EC1-3 in a cranial neural crest explant produces abnormal directional movement. In these experiments, it appeared as though certain regions of the CNC explant were “attracting” other regions of the explant. The preliminary studies described in chapter IV are aimed at answering the question; does EC1-3 attract migrating CNC cells? Here, we generated a Matlab program in order to effectively quantify the amount of directional movement of CNC explants presented with a source of EC1-3. In addition to quantifying cell directionality, this program can also decipher between cells moving with random or directed motion, and measure the velocity of cell migration within certain coordinates. Therefore, this program should be useful other ex vivo studies that require the observation of these features. To conclude, the work presented in this dissertation suggests that the role of cadherin-11 during cranial neural crest migration is predominately based on the adhesive function. In order for CNC migration to proceed, the amount of cadherin-11 mediated cell-cell adhesion is tightly regulated throughout this process. These cell-cell interactions are likely important for “sheet” and “branch” migration where CNC cells maintain a lot of cell-cell cohesion.

Receptor tyrosine kinases (RTKs) are key regulators of critical cellular processes, such as cell cycle, differentiation, proliferation, apoptosis and survival. Mutations, hyperactivity and loss of function of RTKs are responsible for numerous diseases. Because of the therapeutic importance of RTK signaling, intensive studies have been devoted to understanding the signaling mechanisms of RTKs, and the key components in their signaling networks. However, studying the cellular responses to RTK stimulation in a native cellular context is technically challenging. Consequently, many details of RTK signaling kinetics, and the underlying molecular mechanisms of action, remain unclear. The RET receptor tyrosine kinase is important for neuronal cell survival and function, and for the development of the kidneys and nervous system. Gain of function of RET leads to tumor formation, while loss of function in RET’s kinase activity is associated with the developmental kidney defect Hirschsprung’s disease. RET is activated by ligands of glial cell line-derived neurotrophic factor (GDNF) family, which consist of four homologs—GDNF, Neuturin, Artemin (ART) and Persephin. GDNF family ligands activate RET only in the presence of GPI-linked co-receptors (GFRα1–4). Formation of the pentameric ligand/co-receptor2/RET2 complex leads to dimerization of RET and autophosphrylation of its cytoplasmic kinase domain. RET phosphorylation results in the activation of multiple downstream signaling pathways, including the Ras-Raf-MEK-ERK and PI3K-Akt pathways. The ERK and Akt signaling pathways participate in a variety of cellular activities, including cell survival, proliferation, and differentiation. My project addresses the following questions: (1) What are the kinetics of RET activation and deactivation processes after ART stimulation? (2) How is RET activation coupled to the phosphorylation of ERK1/2 and Akt? (3) How does ligand-induced internalization of RET affect RET signaling? (4) How does each step in the RET-Ras-Raf-MEK-ERK cascade quantitatively regulate ERK phosphorylation levels? The results will elucidate the spatial and temporal dynamics of RET signaling upon stimulation by ART, and to determine how downstream signaling is regulated by the amplitude and timing of RET activation. Overall, the thesis aims to advance our understanding of RTK signaling, by establishing methods and principles that can potentially be applied to other RTK systems.

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