Phosphatidylcholine (PC) is an essential component of biological membranes and is synthesized by the CDP-choline pathway under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCT?). Ras transformed cells have increased lipid synthesis; the aim of this study was to determine if upregulation of CCT? was part of this transformed phenotype. Rat intestinal epithelial cell lines (IEC) and three oncogenic H-ras expressing IEC (IEC-Ras) were used to investigate the role of CCT? and phosphatidylcholine (PC) synthesis in resistance to detachment dependant apoptosis, termed anoikis. IEC-Ras have increased CCT? expression within the nucleus. Reduction of CCT? expression with lentiviral short hairpin RNA sensitized IEC-Ras to anoikis and decreased PC degradation, but did not change PC synthesis. Thus, in addition to CCT? being involved in anoikis-resistance in IEC-Ras these data indicate the possibility that it may also have nuclear-specific functions.

The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases, and loss of RAB 25 expression has been reported in a number of breast cancer cases containing H-Ras point mutations, particularly triple negative breast cancers (TNBC), one of the most aggressive subtypes of breast cancer and associated with a poor prognosis. The mechanism involved in the progression of these tumors is poorly understood. In this study, we are trying to understand if loss of RAB25 expression in Human Mammary Epithelial Cell (HMEC) lines co-operates with H-Ras mutations and contributes to tumorigenesis. HMEC were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, catalytic subunit of the telomerase enzyme that permits the cells to exceed the Hayflick Limit and become immortal. We have found that with loss of RAB25 and over expression of mutant H-Ras61L, immortal HMEC undergo transformation. We have looked into the co-operativity between loss of Rab25 and H-Ras61L mutant by in-vitro studies to show their anchorage independent growth and increased ability to migrate. Furthermore, cells express low CD24, high CD44, and very low levels of Claudin indicating that cells acquire stem-like properties upon transformation. Loss of RAB25 and over-expression of H-ras61L resulted in increased expression of transcription markers Snail and Slug that drive these cells to lose E-cadherin and undergo Epithelial Mesenchymal Transition (EMT). This study shows that loss of RAB25 and over-expression of mutant H-Ras can transform HMEC and give rise to mesenchymal stem-like tumors. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker in the prognosis of Claudin-low type of TNBC.

Members of the RAF protein kinase family are key regulators of diverse cellular processes. The need for isoform-specific regulation is reflected by the fact that all RAFs not only display a different degree of activity but also perform isoform-specific functions at diverse cellular compartments. Protein-protein-interactions and phosphorylation events are essential for the signal propagation along the Ras-RAF-MEK-ERK cascade. More than 40 interaction partners of RAF kinases have been described so far. Two of the most important regulators of RAF activity, namely Ras and 14-3-3 proteins, are subject of this work. So far, coupling of RAF with its upstream modulator protein Ras has only been investigated using truncated versions of RAF and regardless of the lipidation status of Ras. We quantitatively analyzed the binding properties of full-length B- and C-RAF to farnesylated H-Ras in presence and absence of membrane lipids. While the isolated Ras-binding domain of RAF exhibit a high binding affinity to both, farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases demonstrate crucial differences in their affinity to Ras. In contrast to C-RAF that requires carboxyterminal farnesylated H-Ras for interaction at the plasma membrane, B-RAF also binds to nonfarnesylated H-Ras in the cytosol. For identification of the potential farnesyl binding site we used several fragments of the regulatory domain of C-RAF and found that the binding of farnesylated H-Ras is considerably increased in the presence of the cysteine-rich domain of RAF. In B-RAF a sequence of 98 amino acids at the extreme N terminus enables binding of Ras independent of its farnesylation status. The deletion of this region altered Ras binding as well as kinase properties of B-RAF to resemble C-RAF. Immunofluorescence studies in mammalian cells revealed essential differences between B- and C-RAF regarding the colocalization with Ras. In conclusion, our data suggest that that B-RAF, in contrast to C-RAF, is also accessible for nonfarnesylated Ras in the cytosolic environment due to its prolonged N terminus. Therefore, the activation of B-RAF may take place both at the plasma membrane and in the cytosolic environment. Furthermore, the interaction of RAF isoforms with Ras at different subcellular sites may also be governed by the complex formation with 14-3-3 proteins. 14-3-3 adapter proteins play a crucial role in the activation of RAF kinases, but so far no information about the selectivity of the seven mammalian isoforms concerning RAF association and activation is available. We analyzed the composition of in vivo RAF/14-3-3 complexes isolated from mammalian cells with mass spectrometry and found that B-RAF associates with a greater variety of 14-3-3 proteins than C- and A-RAF. In vitro binding assays with purified proteins supported this observation since B-RAF showed highest affinity to all seven 14-3-3 isoforms, whereas C-RAF exhibited reduced affinity to some and A-RAF did not bind to the 14-3-3 isoforms epsilon, sigma, and tau. To further examine this isoform specificity we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. By deleting one of the two 14-3-3 isoforms in Saccharomyces cerevisiae we were able to show that homodimeric 14-3-3 proteins are sufficient for functional activation of B- and C-RAF. In this context, the diverging effect of the internal, inhibiting and the activating C-terminal 14-3-3 binding domain in RAF could be demonstrated. Furthermore, we unveil that prohibitin stimulates C-RAF activity by interfering with 14-3-3 at the internal binding site. This region of C-RAF is also target of phosphorylation as part of a negative feedback loop. Using tandem MS we were able to identify so far unknown phosphorylation sites at serines 296 and 301. Phosphorylation of these sites in vivo, mediated by activated ERK, leads to inhibition of C-RAF kinase activity. The relationship of prohibitin interference with 14-3-3 binding and phosphorylation of adjacent sites has to be further elucidated. Taken together, our results provide important new information on the isoform-specific regulation of RAF kinases by differential interaction with Ras and 14-3-3 proteins and shed more light on the complex mechanism of RAF kinase activation. / RAF Protein Kinasen sind essentielle Regulatoren verschiedener zellulärer Prozesse. Unterschiedlich starke Aktivitäten und Lokalisation der drei RAF Isoformen erfordern eine isoform-spezifische Regulation. Der Einfluss von Protein-Protein Interaktionen und Phosphorylierungen ist dabei mitentscheidend für die Signalweiterleitung entlang der Ras-RAF-MEK-ERK Kaskade. Mehr als 40 Interaktionspartner der RAF Kinasen wurden bereits beschrieben von denen zwei der wichtigsten, Ras und 14-3-3 Proteine, Gegenstand der vorliegenden Arbeit sind. Die Interaktion von RAF mit seinem vorgeschaltetem Modulatorprotein Ras wurde bislang nur mit verkürzten RAF-Proteinen und ohne Rücksicht auf den Lipidierungsgrad von Ras untersucht. Wir haben die Bindeeigenschaften von B- und C-RAF in voller, nativer Länge zu farnesyliertem H-Ras in Gegenwart und Abwesenheit von Membranlipiden quantifiziert. Während die isolierte Ras-Bindungsdomäne eine hohe Affinität sowohl zu farnesyliertem als auch nicht-farnesyliertem H-Ras aufweist, zeigen die RAF Proteine in voller Länge entscheidende Unterschiede in ihrem Bindeverhalten zu Ras. C-RAF benötigt für eine effiziente Interaktion mit H-Ras dessen C-terminale Farnesylgruppe, wobei B-RAF auch an nicht-farnesyliertes H-Ras im Cytosol bindet. Um die verantwortliche Farnesylbinderegion zu identifizieren haben wir verschiedene Fragmente der regulatorischen Domäne von C-RAF eingesetzt. Dadurch konnten wir zeigen, dass die Affinität zu farnesyliertem Ras in Gegenwart der sogenannten Cystein-reichen Domäne von RAF beträchtlich erhöht war. In B-RAF ist eine Sequenz von 98 Aminosäuren am N-Terminus verantwortlich für die Ras-Bindung unabhängig von dessen Farnesylierungszustand. Die Deletion dieser Sequenz von B-RAF veränderte die Ras-Bindungseigenschaften sowie die Kinaseaktivität vergleichbar mit C-RAF. Durch Immunfluoreszenzversuche in Säugerzellen konnten darüber hinaus Unterschiede in der Kolokalisation von B- und C-RAF mit Ras beobachtet werden. Zusammenfassend deuten unsere Ergebnisse darauf hin, dass B-RAF, im Gegensatz zu C-RAF, aufgrund seines verlängerten N-Terminus in der Lage ist bereits im Cytosol auch mit unfarnesyliertem Ras zu interagieren, wodurch die Aktivierung von B-RAF sowohl im Cytosol als auch an der Plasmamenbran erfolgen kann. Die Interaktion der RAF-Isoformen mit Ras in unterschiedlichen zellulären Kompartimenten kann aber auch durch die Komplexbildung mit 14-3-3 Proteinen beeinflusst werden. Die 14-3-3 Adapter Proteine spielen eine entscheidende Rolle im Aktivierungszyklus der RAF Proteine. Bislang waren jedoch keine Details bezüglich der Selektivität der sieben 14-3-3 Isoformen aus Säugerzellen hinsichtlich der Assoziation mit und Aktivierung der RAF Kinasen bekannt. Wir haben RAF/14-3-3 Komplexe aus Säugerzellen isoliert und durch Massenspektrometrie analysiert. Dadurch konnten wir zeigen, dass B-RAF mit einer größeren Vielfalt an 14-3-3 Isoformen bindet als C- und A-RAF. In vitro Bindungsversuche mit gereinigten Proteinen bestätigten die höhere Affinität von B-RAF zu allen sieben Säuger-14-3-3 Proteinen. C-RAF dagegen zeigte eine deutlich reduzierte Affinität, während für A-RAF keine Bindung zu den 14-3-3 Isoformen epsilon, sigma, und tau festgestellt wurde. Um diese Isoformspezifität weiter aufzuklären haben wir untersucht, ob sowohl Homo- als auch Heterodimere von 14-3-3 in der Lage sind die RAF-Signaltransduktion zu beeinflussen. Durch die Deletion einer der beiden 14-3-3 Isoformen aus Saccharomyces cerevisiae konnten wir zeigen, dass bereits ein 14-3-3 Homodimer für die korrekte Aktivierung von B- und C-RAF ausreichend ist. In diesem Zusammenhang konnte auch die Rolle der internen, inhibierenden 14-3-3 Bindestelle in RAF gegenüber der C-terminalen, aktivierenden Stelle dargelegt werden. Zusätzlich zeigen wir, dass Prohibitin seinen aktivierenden Einfluss gegenüber C-RAF durch die Beeinträchtigung der 14-3-3 Bindung an der internen Stelle in RAF ausübt. Diese Region in C-RAF ist das Ziel von Phosphorylierungen im Zuge eines negativen Rückkopplungsmechanismus. Durch den Einsatz von Tandem-Massenspektrometrie konnten wir bislang unbekannte Phosphorylierungsstellen an den Serinen 296 und 301 identifizieren deren ERK-vermittelte Phosphorylierung in vivo eine Inaktivierung der C-RAF bewirkt. Der Zusammenhang zwischen der Behinderung der 14-3-3 Anlagerung durch Prohibitin und die Phosphorylierung in unmittelbarer Nachbarschaft bedarf weiterer Untersuchungen. Zusammengefasst liefern unsere Ergebnisse wichtige Informationen bezüglich der isoform-spezifischen Regulation der RAF Kinasen durch die Interaktion mit Ras und 14-3-3 Proteinen und helfen die komplexen Mechanismen der RAF Aktivierung weiter aufzuklären.

The ras genes, including Harvey ras (H-ras) and Kirsten ras (K-ras), were among the first oncogenes discovered, and are the most commonly mutated oncogenes in human cancer. The H-ras and K-ras proteins are 85% identical and share considerable functional overlap. However, there is increasing evidence for functional differences between the two proteins that may impart different properties to tumors arising from mutations in these two genes. To study the functional differences between H-ras and K-ras in an in vivo setting, we used two different transgenic mouse tumor models, MMTV-H-ras and MMTV-K-ras mice. The MMTV-H-ras mice were originally developed in Dr. Leder's lab and have been well characterized with regard to tumor properties. We created a similar line of transgenic mice expressing mutant K-ras (G12V) under the control of the MMTV promoter. Female mice of both lines develop primarily mammary tumors. We compared differences between the H-ras and K-ras lines with regard to age of tumor onset, rate of tumor growth, and rates of tumor proliferation and apoptosis. The tumors were also characterized by microarray analysis to look for genes that are differentially expressed in the two tumor types. Finally, the response of tumors to two common chemotherapeutic agents, doxorubicin and taxol, was also measured. We found that tumors in the MMTV-H-ras and MMTV-K-ras mice were similar with respect to several tumor properties, including age of onset, histopathology, and proliferation and apoptotic indices. While tumors from mice of these two genotypes clustered separately in an unsupervised analysis of gene expression profiles, the differentially expressed genes did not fall within any well-defined signaling pathways. However, drug studies indicated differences in response to doxorubicin between the two isoforms, with H-ras tumors responding better than K-ras tumors. In conclusion, our studies point to specific differences between H-ras and K-ras that may represent novel signaling pathways not currently known to be regulated by Ras. In spite of the few differences in properties of tumors arising from H-ras and K-ras mutation, there might be differences in response to chemotherapeutic agents that could have clinical significance.

L'objectif de cette thèse visait à améliorer la compréhension des mécanismes présidant à<br />l'activation du moteur moléculaire assurant l'entrée en division des cellules eucaryotes : le<br />MPF (M-Phase promoting Factor). Pour cela, le modèle d'étude sélectionné a été les divisions<br />méiotiques de l'ovocyte de Xénope. Les ovocytes de Xénope sont naturellement bloqués en<br />prophase de méiose I. En réponse à la progestérone, ils reprennent la méiose et se bloquent à<br />nouveau en métaphase II en attente de la fécondation. Ce processus, appelé maturation<br />méiotique, est sous le contrôle du complexe Cdc2-Cycline B, facteur universel de division des<br />cellules eucaryotes. Nous nous sommes intéressés à l'étude des mécanismes régulant l'activité<br />de la kinase Cdc2 au cours de la maturation méiotique. Dans un premier temps, nous avons<br />étudié la régulation de Myt1, une kinase de la famille Wee1. Ces kinases catalysent une<br />phosphorylation inhibitrice sur la protéine Cdc2 et sont donc responsables du maintien du<br />MPF sous une forme inactive pendant la phase G2 du cycle cellulaire. L'activation du MPF<br />repose sur la conversion du stock de pré-MPF inactif en stock de MPF actif, suite à la<br />déphosphorylation activatrice de Cdc2 par la phosphatase Cdc25, et à l'inhibition de Myt1.<br />Nous avons montré que l'activité de Cdc2 était nécessaire à l'inhibition de Myt1 et que deux<br />kinases, p90Rsk et Plx1, sont recrutées l'une ou l'autre pour contribuer à cette inhibition.<br />Dans un deuxième temps, nous sommes intéressés à l'implication de la protéine H-Ras lors de<br />la reprise de la méiose. Nous avons montré que dans l'ovocyte de Xénope, l'injection de HRas<br />induit la reprise de la méiose par le recrutement d'une PI3 kinase particulière. Cette voie,<br />bien que présente et activable dans l'ovocyte, n'est pas recrutée in vivo par la progestérone en<br />conditions normales. L'ovocyte est donc équipé de plusieurs voies de signalisation<br />fonctionnellement redondantes, susceptibles de conduire à l'activation du MPF, qui peuvent<br />être recrutées dans des conditions pathologiques pour assurer la reprise de la méiose quand les<br />effecteurs normaux ne sont pas disponibles.

No description available.

<p>Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays.</p><p>The role of the signaling protein H-Ras in FGF-2-induced <i>in vitro</i> angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. <i>In vitro</i> angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-α (TNF-α). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation.</p><p>In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation. </p>

Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays. The role of the signaling protein H-Ras in FGF-2-induced in vitro angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. In vitro angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-α (TNF-α). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation. In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation.