Cathie Pfleger - US grants

DESCRIPTION (provided by applicant): Signaling through the well-known oncogene Ras can promote proliferation, cell survival, and differentiation and is implicated in cancer. Mutations that increase signaling through this pathway are found frequently in cancer and also occur in developmental disorders such as Noonan's Syndrome. Thus, it is vital to understand how Ras is regulated. Much attention has focused on understanding activation of this pathway at upstream steps, such as the binding of ligands to Receptor Tyrosine Kinases (RTKs), which act through other proteins to recruit and activate Ras. Recently, modification of Ras by a process called ubiquitination has been reported in mammalian cells. My research group now has data confirming the ubiquitination of Ras using Drosophila (fruit flies). This suggests that this newly discovered regulation of Ras is highly conserved. Moreover, using Drosophila (fruit flies) we generated compelling evidence that the ubiquitination of Ras has profound effects on growth, proliferation and cell survival. The powerful system of Drosophila genetics and the well-established paradigm of studying Ras in Drosophila make this an excellent system to address and characterize the significance of Ras ubiquitination in a multi-cellular context. Furthermore, an in vivo Drosophila approach has the potential to make significant and unique discoveries that would not be possible in other systems. The goal of this proposal is to use Drosophila to understand the biological role of Ras ubiquitination. Our hypothesis is that ubiquitination of Ras restricts its ability to promote growth and proliferation. Our hypothesis is based on our Drosophila findings that (1) Drosophila Ras is ubiquitinated, (2) impairing ubiquitination promotes increased signaling through Ras, and (3) impairing ubiquitination promotes Ras-dependent overgrowth, increased, proliferation, and cell death resistance that appear independent of steps upstream of Ras. The novelty of our findings is that Ras may be a distinct step in the pathway whose ubiquitination is crucial to ensure proper control of growth and proliferation. My research group will exploit our expertise in studying the ubiquitin pathway and in utilizing Drosophila to most efficiently attack this problem. The proposed studies perfectly complement ongoing molecular and tissue culture studies being performed elsewhere. In the short term, our studies could establish the biological significance of this novel regulation of Ras. Over the longer term, our work could contribute tremendously towards a better understanding of the Ras oncogene and may even identify therapeutic targets. Therefore, we believe that these studies could have profound implications for cancer research. We propose research according to the following Aims: Specific Aim 1: Establish the biological significance of Ras ubiquitination. Specific Aim 2: Examine Ras ubiquitination in vivo. Specific Aim 3: Identify and characterize the Ras E3. PUBLIC HEALTH RELEVANCE: Mutations that activate a protein called Ras or that increase signaling through the Ras pathway are frequently found in developmental disorders and in cancer. Therefore, it is crucial that we understand how Ras signaling is regulated. The proposed studies will investigate a novel regulation of Ras by a process called "ubiquitination" and could contribute tremendously towards a better understanding of Ras and may have profound implications for cancer research.

When a single fertilized cell develops into an intricately patterned organism with multiple organs (such as the heart and liver), those organs grow to predetermined sizes; when problems occur that cause injury, growth retardation, or increased growth, organs still reach and then stop at their predetermined sizes. This is because of a process called the "organ size checkpoint". Despite years of study, how the organ size checkpoint works remains an important, fundamental question in biology. Utilizing the powerful genetic system of the fruit fly, several research teams discovered that a group of genes called the Hippo pathway work together as a "foreperson" or "overseer" of the organ size checkpoint. The Hippo pathway coordinates when cells in an organ grow, divide, or die by delegating other genes to carry out these tasks by turning these other genes on or off. The proposed studies will use cultured cells, biochemistry, and fruit fly genetics to identify these important other genes controlled by the Hippo pathway. Identifying these genes will advance our understanding of the fundamental regulation of cell division, cell growth, and cell death during development and will shed tremendous light on how organs grow to and are maintained at the appropriate size.

The project will provide high school, undergraduate, and graduate students with experiences catered to their diverse backgrounds and experience levels: younger students will learn to work and think independently while collaborating with advanced students who engage in detailed, hypothesis-driven experiments. Working and communicating as a team will give younger students a valuable foundation for a career in science and will help graduate students develop mentoring skills; therefore, this work will have a broader impact of training young scientists and instilling leadership skills they need to drive the next generation of scientific breakthroughs.

SUMMARY Ras signaling promotes proliferation, cell survival, and differentiation and is implicated in cancer. To devise effective therapeutics, it is vital to understand how Ras is regulated and how to block its activity. My lab discovered that modifying Ras by attaching the small protein ubiquitin restricts both wild-type and oncogenic forms of Ras. Impairing Ras ubiquitination in vivo in Drosophila led to striking effects on cell proliferation, cell survival, developmental patterning, and organismal longevity, reflecting a role for Ras ubiquitination in development, tumor suppression, and survival. We identified the Ras E3 (the ubiquitin ligase that adds ubiquitin to Ras) as Rabex-5 (also called RabGEF1). Intriguingly, we discovered that Rabex-5 also regulates the Notch oncogene. The power of Drosophila genetics and the well-established paradigm of studying Ras in Drosophila make this system ideally-suited to characterize these phenomena in a multi-cellular context. The goal of this proposal is to use in vivo Drosophila studies and in vitro biochemical analysis to elucidate the biological significance and molecular mechanisms of Ubiquitin Pathway regulation of Ras and Notch signaling. Defining the molecular mechanism(s) of Ras ubiquitination will yield key insights into fundamental biology and will reveal novel points of therapeutic entry in cancer. We made initial observations regarding spatial and temporal regulation of Ras ubiquitination. Consistent with data that Rabex-5 is lost or decreased in various cancers, our loss-of-function studies unambigiously indicate a tumor suppressor role for Rabex-5 by inhibiting Ras and Notch signaling. We hypothesize that Rabex-5 restricts Ras and Notch and acts in tumor suppression via its E3 domain. By complementing in vivo Drosophila work with in vitro biochemistry, we are uniquely positioned to make significant advances into conserved, fundamental mechanisms of regulation and coordination of Ras and Notch oncogenic signaling.

Alcohol consumption can have life-threatening effects on human health; in fact, the overall disease burden from alcohol is estimated to exceed 3% of deaths throughout the world. The consequences of alcohol are particularly severe in the liver where alcohol is metabolized and leads to Alcoholic Liver Disease (ALD). ALD occurs as a variety of conditions including fatty liver (steatosis), hepatitis, fibrosis, and hepatocellular carcinoma (HCC). Other behaviors with adverse effects on health, such as smoking, often coincide with alcohol consumption, patients under-report their consumption of alcohol, and there is substantial genetic variation across the population; therefore, elucidating the mechanisms underlying ALD in patients presents a number of significant challenges. To overcome these challenges in ALD research, we developed a Drosophila (fruit fly) model of ALD using the Drosophila organ most similar to the mammalian liver, a bipartite organ of the ?fat body? and the ?oenocytes.? Strikingly, the fat body significantly increases in size (organomegaly) in larvae exposed to alcohol, reminiscent of mammalian ethanol-induced hepatomegaly. Existing animal models for ALD have some advantages but also face certain limitations. Drosophila recapitulate many phenomena relevant to disease, and this model system has been used effectively to model diverse conditions from autism to cancer. Drosophila represent an ideal system to explore effects of genetic susceptibility, including germline or somatic mutations in specific tissues, combined with environmental exposure such as exposure to alcohol and other forms of oxidative stress. Although our Drosophila ALD model also faces limitations, it bypasses some inherent limitations of existing animal models and brings the power of Drosophila genetics to the study of ALD, namely (1) 10-day generation time, (2) the ease of performing a large number of parallel genetic interaction studies, (3) the utility of descriptive phenotypic analysis, (4) the ability to address the functional relevance of candidate molecules in physiological setting, and (5) the wealth of genetic tools and reagents. Thus, our Drosophila ALD model provides an extremely practical, rapid, efficient, and cost- effective system for discovery and for developing meaningful mechanistic hypotheses to then address in vertebrate models. Establishing the Drosophila fat body and oenocytes as a model to study ALD will be a tremendous resource and make a significant impact on the field. Flies are also an excellent in vivo system capable of simultaneously evaluating chemical compound stability, bioavailability, toxicity, and efficacy in modifying disease-relevance phenotypes. In summary, our Drosophila ALD model is a valuable resource that complements existing ALD model systems to significantly advance our understanding of the etiology of ALD and to serve as a platform for drug discovery.

SUMMARY Eukaryotic signaling pathways translate external cues in specific contexts into diverse biological outputs. Proper timing and duration of a signaling response requires careful regulation of stability, trafficking, activation, and deactivation of multiple components within a network. This overall coordination can involve positive feedback circuits to amplify signaling and/or negative feedback circuits to constrain signaling. The Ras signaling pathway is crucial in development to regulate proliferation, cell survival, cell fate, and patterning. Mutations in Ras or other components of the pathway that lead to increased Ras signaling cause developmental disorderes collectively known as ?Rasopathies? such as Noonan's syndrome or Neurofibromatosis Type 1 (NF1). Using Drosophila, we discovered that Rabex-5 (an E3) downregulates Ras by promoting its mono- and di-ubiquitination. Specifically, we discovered that impairing Ras ubiquitination in vivo in Drosophila led to striking effects on cell proliferation, cell survival, developmental patterning, and organismal longevity, reflecting a role for Ras ubiquitination in development, tumor suppression, and survival. The power of Drosophila genetics and the well-established paradigm of studying Ras in Drosophila make this system ideally suited to characterize this phenomenon in a multi-cellular context. We more recently discovered that Rabex-5 targeting of Ras requires an N-terminal tyrosine in Ras, Tyrosine 4 (Y4). We hypothesize that phosphorylation of this tyrosine directs targeting of Ras by Rabex-5. Our hypothesis predicts that the kinase(s) and phosphatase(s) that regulate modification of Y4 are crucial regulators of Ras signaling. The goal of our application is to elucidate the role of tyrosine phosphorylation of Ras to direct its inhibitory Rabex-5- mediated ubiquitination and maintain pathway homeostasis. Specifically, we propose to further characterize the biological role of Y4 phosphorylation in Ras ubiquitination, to identify the Ras Y4 kinase(s) and phosphatase(s), and to characterize their roles in regulating Ras in development.